RNAi modulation of TGF-beta and therapeutic uses thereof

ABSTRACT

The present invention concerns methods of treatment using transforming growth factor beta (TGF-beta) modulators. More specifically, the invention concerns methods of treating disorders associated with undesirable TGF-beta signaling, by administering short interfering RNA which down-regulate the expression of TGF-beta, and agents useful therein.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/782,829, filed Mar. 16, 2006, which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention concerns methods of treatment using transforminggrowth factor beta (TGF-beta) modulators. More specifically, theinvention concerns methods of treating disorders associated withundesirable TGF-beta signaling, by administering short interfering RNAwhich down-regulate the expression of TGF-beta, and agents usefultherein.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi has beensuggested as a method of developing a new class of therapeutic agents.However, to date, these have remained mostly as suggestions with nodemonstrate proof that RNAi can be used therapeutically.

Transforming growth factor-beta (TGF-beta) isoforms (−1, −2, & −3) arepotent cytokines that act in autocrine and paracrine fashion to effect abroad spectrum of biological processes, including proliferation,differentiation, apoptosis, and extracellular matrix production.TGF-beta exerts its biological effects via binding to TGF-beta receptors(types I, II, & III). The local over-expression of TGF-beta in thekidney, liver, or lung mediates the pathophysiology observed in diabeticnephropathy, chronic liver disease, and pulmonary fibrosis,respectively. TGF-beta1 is up-regulated in patients with diabeticnephropathy (for both type 1 or type 2 diabetes) and in animal models ofthe disease. Antibodies to TGF-beta have been shown to prevent diseasein a mouse genetic model of type 2 diabetes (db/db mouse) and antisensetargeting a conserved sequence in TGF-beta RNA has been shown to preventdisease in a streptozotocin-diabetic mouse model.

About 100,000 diabetic patients per year are treated for kidney diseasein U.S. with health costs of $5.1 billion per year in the U.S. Diabeticnephropathy is the leading cause of end-stage renal disease in theindustrialized world. Almost 40% of all new patients with renal failureadmitted to renal replacement programs in the U.S have diabetic kidneydisease. Albuminuria, proteinuria, serum creatinine, as well ascirculating TGF-beta1 plasma levels, can be used as markers for efficacydetermination in therapeutic evaluation.

Liver disease affects all age groups and both genders. The condition canbe acute or chronic. The major causes of liver diseases in the UnitedStates are viruses (hepatitis A, B, C) and alcohol abuse. However,congenital, autoimmune and drug-induced causes are also significantcontributors to the origin of liver diseases. Liver diseases disturbhepatic functions as a result of destruction of functional liver tissueand development of fibrosis (scarring), which blocks blood flow throughthe liver and causes portal hypertension (pressure in the portal vein).Blood flow then seeks an alternative route, leading to dilated swollenveins (varices) in the esophagus that may hemorrhage. Liver disease andportal hypertension also alter kidney function by causing retention ofsalt and water (ascites), and can induce renal failure (hepatorenalsyndrome) and altered mental state (coma). The primary cytokine involvedin tissue scaring in chronic liver disease is TGF-beta1. Recently it hasbeen shown in a rat model of liver fibrosis that blocking TGF-beta1 canlead to improvement in liver histology. Further, in chronic Hepatitis Cpatients who have responded to interferon alpha, decreased levels ofTGF-beta1 are associated with improvements in liver fibrosis.

TGF-beta1 has also been implicated in Idiopathic Pulmonary Fibrosis.Similar to liver disease, the mechanisms of action of TGF-beta1 inpulmonary fibrosis involves both induction and inhibition of thedegradation of extracellular matrix proteins, leading to the formationof scar tissue. TGF-beta1 mediated cell signaling, primarily at thelocal site of connective tissue, is anabolic and leads to pulmonaryfibrosis and angiogenesis, strongly indicating that TGF-beta1 may beinvolved in the repair of tissue injury caused by burns, trauma, orsurgery. Pulmonary fibrosis, also called Interstitial Lung Disease(ILD), is a broad category of lung diseases that includes more than 130disorders which are characterized by scarring of the lungs. ILD accountsfor 15% of the cases seen by pulmonologists. Some of the interstitiallung disorders include: Idiopathic pulmonary fibrosis, Hypersensitivitypneumonitis, Sarcoidosis, Eosinophilic granuloma, Wegener'sgranulomatosis, Idiopathic pulmonary hemosiderosis, and Bronchiolitisobliterans. In ILD, scarring or fibrosis occurs as a result of either aninjury or an autoimmune process. Approximately 70% of ILD have noidentifiable cause and are therefore termed “idiopathic pulmonaryfibrosis.” Some of the known causes include occupational andenvironmental exposure, dust (silica, hard metal dusts), organic dust(bacteria, animal proteins), gases and fumes, drugs, poisons,chemotherapy medications, radiation therapy, infections, connectivetissue disease, systemic lupus erythematosus, and rheumatoid arthritis.In its severest form, ILD can lead to death, which is often caused byrespiratory failure due to hypoxemia, right-heart failure, heart attack,stroke, blood clot (embolism) in the lungs, or lung infection brought onby the disease.

The use of small interfering nucleic acid molecules targetingtransforming growth factor beta (TGF-beta) genes provides a class ofnovel therapeutic agents that can be used in the treatment of variousdiseases and conditions associated with undesired TGF-beta signaling,including diabetic nephropathy, chronic liver disease, pulmonaryfibrosis, hematopoietic reconstitution, and any other inflammatory,respiratory, autoimmune, and/or proliferative disease, condition, ortrait that responds to the level of TGF-beta in a cell, subject ororganism, and particularly idiopathic pulmonary fibrosis.

SUMMARY

The present invention is based on the in vitro demonstration thatTGF-beta expression can be inhibited by iRNA agents, and theidentification of potent iRNA agents from the TGF-beta gene that canreduce RNA levels and protein levels of TGF-beta in cells, andparticularly in an organism. Based on these findings, the presentinvention provides specific compositions and methods that are useful inreducing TGF-beta mRNA levels and TGF-beta protein levels in cells invitro as well as in a subject in vivo, e.g., a mammal, such as a human.These compositions are particularly useful in the manufacturing ofpharmaceutical compositions for the treatment of, and ultimately inmethods to treat, subjects having or at risk of developing a disease ordisorder associated with undesired TGF-beta signaling such as, forexample, idiopathic pulmonary fibrosis.

The present invention specifically provides methods of reducing thelevels of a TGF-beta mRNA in a cell of a subject, or of TGF-beta proteinsecreted by a cell of a subject, comprising the step of administering aniRNA agent to said subject, wherein the iRNA agent comprises anantisense strand consisting of 15 to 30 nucleotides and having at least15 or more contiguous nucleotides complementary to a mammalian TGF-betamRNA, and a sense strand consisting of 15 to 30 nucleotides and havingat least 15 or more contiguous nucleotides complementary to saidantisense strand. Said iRNA agent may mediate the cleavage of a TGF-betamRNA within the target sequence of an iRNA agent selected from the groupof: AL-DP-6837 to AL-DP-6864 and AD-14419 to AD-14638. In oneembodiment, said iRNA agent comprises 15 or more contiguous nucleotidesfrom an iRNA agent selected from the group of: AL-DP-6837 to AL-DP-6864,AL-DP-6140 to AL-DP-6151, AL-DP-6262 to AL-DP-6277 and AD-14419 toAD-14638. In a further embodiment, said iRNA agent is one of the iRNAagents selected from the group of: AL-DP-6837 to AL-DP-6864, AL-DP-6140to AL-DP-6151, AL-DP-6262 to AL-DP-6277 and AD-14419 to AD-14638. Infurther embodiments, the iRNA agents mediates the cleavage of a TGF-betamRNA within the target sequence of an iRNA agent, comprises 15 or morecontiguous nucleotides from an iRNA agent, or is an iRNA agent, selectedfrom one of the following groups: the 20%-group, the 30%-group, the40%-group, the 50%-group, the 60%-group, the 70%-group, the 80%-group,or the 90%-group, as defined below. Said iRNA agent may be administeredto a subject, wherein administration may comprise pulmonaryadministration. As a result of the inventive method, the level ofTGF-beta protein and/or TGF-beta mRNA may be reduced by at least 20%.

In another aspect, the invention provides isolated iRNA agents,comprising an antisense strand consisting of 15 to 30 nucleotides andhaving at least 15 or more contiguous nucleotides complementary to amammalian TGF-beta mRNA, and a sense strand consisting of 15 to 30nucleotides and having at least 15 or more contiguous nucleotidescomplementary to said antisense strand. In one embodiment, said iRNAagent mediates the cleavage of a TGF-beta mRNA within the targetsequence of an iRNA agent selected from one of the following groups: thegroup of AL-DP-6837 to AL-DP-6864 and AD-14419 to AD-14638, the20%-group, the 30%-group, the 40%-group, the 50%-group, the 60%-group,the 70%-group, the 80%-group, or the 90%-group, as defined below. In afurther embodiment, said iRNA agent comprises 15 or more contiguousnucleotides from an iRNA agent selected from one of the followinggroups: the group of AL-DP-6837 to AL-DP-6864, AL-DP-6140 to AL-DP-6151,AL-DP-6262 to AL-DP-6277 and AD-14419 to AD-14638 the 20%-group, the30%-group, the 40%-group, the 50%-group, the 60%-group, the 70%-group,the 80%-group, or the 90%-group, as defined below. More specifically,said iRNA agent may be an iRNA agent selected from one of the followinggroups of: AL-DP-6837 to AL-DP-6864, AL-DP-6140 to AL-DP-6151,AL-DP-6262 to AL-DP-6277 and AD-14419 to AD-14638; the 20%-group, the30%-group, the 40%-group, the 50%-group, the 60%-group, the 70%-group,the 80%-group, or the 90%-group, as defined below. The iRNA agent mayfurther comprise a non-nucleotide moiety. Furthermore, the sense and/orantisense strand may be stabilized against nucleolytic degradation. TheiRNA agent may further comprise at least one 3′-overhang, wherein said3′-overhang comprises from 1 to 6 nucleotides. Also, the iRNA agent maycomprise a phosphorothioate at the first internucleotide linkage at the5′ end of the antisense and/or sense sequence, and optionally a furtherphosphorothioate at the first internucleotide linkage at the 3′ end ofthe antisense and/or sense sequence. The iRNA agent may comprise a2′-modified nucleotide, preferably selected from the group consistingof: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O—NMA).

In another aspect, the instant invention features a method of reducingthe levels of a TGF-beta mRNA in a cell, or of TGF-beta protein secretedby a cell, comprising contacting the cell with an iRNA agent of theinvention.

In yet another aspect, the instant invention provides a vector encodingan iRNA agent of the invention.

In yet another aspect, the instant invention provides a cell comprisingan iRNA agent or a vector of the invention.

In yet another aspect, the a method of making an iRNA agent of theinvention is provided, the method comprising the synthesis of the iRNAagent, wherein the sense and antisense strands comprise at least onemodification that stabilizes the iRNA agent against nucleolyticdegradation.

In yet another aspect, the instant invention provides a pharmaceuticalcomposition comprising an iRNA agent of the invention and apharmaceutically acceptable carrier.

In yet another aspect, the instant invention provides a method oftreating a human diagnosed as having or at risk for developing a diseaseor disorder associated with undesired TGF-beta signaling, comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of an iRNA agent of the invention. In one embodiment,the human is diagnosed as having or at risk for having idiopathicpulmonary fibrosis, diabetic nephropathy or chronic liver disease.

Table 1 provides exemplary iRNA agents of the invention.

TABLE 1 Oligonucleotide sequences of siRNAs specific for TGF-betaPosition of target sequence in Duplex SEQ ID SEQ ID NM_000660.3identifier sense strand sequence¹ NO: antisense strand sequence¹ NO:1186-1208 AL-DP-6837 ggucacccgcgugcuaaugTT 1 cauuagcacgcgggugaccTT 21186-1208 AL-DP-6140 ggumcmacmcmcmgcmgumgcmumaaumgTT 3cmauumagcmacgcgggugaccTT 4 1184-1206 AL-DP-6838 gaggucacccgcgugcuaaTT 5uuagcacgcgggugaccucTT 6 1184-1206 AL-DP-6141gaggumcmacmcmcmgcmgumgcmumaaTT 7 uumagcmacgcgggugaccucTT 8 1112-1134AL-DP-6839 agcacccgcgaccggguggTT 9 ccacccggucgcgggugcuTT 10 1112-1134AL-DP-6142 agcmacmcmcmgcmgacmcmgggumggTT 11 ccmacccggucgcgggugcuTT 121797-1819 AL-DP-6840 uccacgagcccaagggcuaTT 13 uagcccuugggcucguggaTT 141797-1819 AL-DP-6143 umcmcmacmgagcmcmcmaagggcmumaTT 15umagcccuugggcucguggaTT 16 444-466 AL-DP-6841 auuccggaccagcccucggTT 17ccgagggcugguccggaauTT 18 444-466 AL-DP-6144aumumcmcmggacmcmagcmcmcmumcmggTT 19 ccgagggcugguccggaauTT 20 1185-1207AL-DP-6842 aggucacccgcgugcuaauTT 21 auuagcacgcgggugaccuTT 22 1185-1207AL-DP-6145 aggumcmacmcmcmgcmgumgcmumaaumTT 23 auumagcmacgcgggugaccuTT 24445-467 AL-DP-6843 uuccggaccagcccucgggTT 25 cccgagggcugguccggaaTT 26445-467 AL-DP-6146 umumcmcmggacmcmagcmcmcmumcmgggTT 27cccgagggcugguccggaaTT 28 1104-1126 AL-DP-6844 uguacaacagcacccgcgaTT 29ucgcgggugcuguuguacaTT 30 1104-1126 AL-DP-6147umgumacmaacmagcmacmcmcmgcmgaTT 31 ucgcgggugcuguugumacmaTT 32 1189-1211AL-DP-6845 cacccgcgugcuaauggugTT 33 caccauuagcacgcgggugTT 34 1189-1211AL-DP-6148 cmacmcmcmgcmgumgcmumaaumggumgTT 35 cmaccmauumagcmacgcgggugTT36 446-468 AL-DP-6846 uccggaccagcccucgggaTT 37 ucccgagggcugguccggaTT 38446-468 AL-DP-6149 umcmcmggacmcmagcmcmcmumcmgggaTT 39ucccgagggcugguccggaTT 40 1787-1809 AL-DP-6847 uggaaguggauccacgagcTT 41gcucguggauccacuuccaTT 42 1787-1809 AL-DP-6150umggaagumggaumcmcmacmgagcmTT 43 gcucguggauccmacuuccmaTT 44 1806-1828AL-DP-6848 ccaagggcuaccaugccaaTT 45 uuggcaugguagcccuuggTT 46 1806-1828AL-DP-6151 cmcmaagggcmumacmcmaumgcmcmaaTT 47 uuggcmauggumagcccuuggTT 481187-1209 AL-DP-6849 gucacccgcgugcuaauggTT 49 ccauuagcacgcgggugacTT 501187-1209 AL-DP-6262 gumcmacmcmcmgcmgumgcmumaaumggTT 51ccmauumagcmacgcgggugacTT 52 1106-1128 AL-DP-6850 uacaacagcacccgcgaccTT53 ggucgcgggugcuguuguaTT 54 1106-1128 AL-DP-6263umacmaacmagcmacmcmcmgcmgacmcmTT 55 ggucgcgggugcuguugumaTT 56 1804-1826AL-DP-6851 gcccaagggcuaccaugccTT 57 ggcaugguagcccuugggcTT 58 1804-1826AL-DP-6264 gcmcmcmaagggcmumacmcmaumgcmcmTT 59 ggcmauggumagcccuugggcTT 601790-1812 AL-DP-6852 aaguggauccacgagcccaTT 61 ugggcucguggauccacuuTT 621790-1812 AL-DP-6265 aagumggaumcmcmacmgagcmcmcmaTT 63ugggcucguggauccmacuuTT 64 239-261 AL-DP-6853 gggaggagggacgagcuggTT 65ccagcucgucccuccucccTT 66 239-261 AL-DP-6266 gggaggagggacmgagcmumggTT 67ccmagcucgucccuccucccTT 68 1107-1129 AL-DP-6854 acaacagcacccgcgaccgTT 69cggucgcgggugcuguuguTT 70 1107-1129 AL-DP-6267acmaacmagcmacmcmcmgcmgacmcmgTT 71 cggucgcgggugcuguuguTT 72 1807-1829AL-DP-6855 caagggcuaccaugccaacTT 73 guuggcaugguagcccuugTT 74 1807-1829AL-DP-6268 cmaagggcmumacmcmaumgcmcmaacmTT 75 guuggcmauggumagcccuugTT 76447-469 AL-DP-6856 ccggaccagcccucgggagTT 77 cucccgagggcugguccggTT 78447-469 AL-DP-6269 cmcmggacmcmagcmcmcmumcmgggagTT 79cucccgagggcugguccggTT 80 1711-1733 AL-DP-6857 caacuauugcuucagcuccTT 81ggagcugaagcaauaguugTT 82 1711-1733 AL-DP-6270cmaacmumaumumgcmumummagcmumcmcmTT 83 ggagcugaagcmaaumaguugTT 841748-1770 AL-DP-6858 gugcggcagcuguacauugTT 85 caauguacagcugccgcacTT 861748-1770 AL-DP-6271 gumgcmggcmagcmumgumacmaumumgTT 87cmaaugumacmagcugccgcmacTT 88 1788-1810 AL-DP-6859 ggaaguggauccacgagccTT89 ggcucguggauccacuuccTT 90 1788-1810 AL-DP-6272ggaagumggaumcmcmacmgagcmcmTT 91 ggcucguggauccmacuuccTT 92 1789-1811AL-DP-6860 gaaguggauccacgagcccTT 93 gggcucguggauccacuucTT 94 1789-1811AL-DP-6273 gaagumggaumcmcmacmgagcmcmcmTT 95 gggcucguggauccmacuucTT 961798-1820 AL-DP-6861 ccacgagcccaagggcuacTT 97 guagcccuugggcucguggTT 981798-1820 AL-DP-6274 cmcmacmgagcmcmcmaagggcmumacmTT 99gumagcccuugggcucguggTT 100 1802-1824 AL-DP-6862 gagcccaagggcuaccaugTT101 caugguagcccuugggcucTT 102 1802-1824 AL-DP-6275gagcmcmcmaagggcmumacmcmaumgTT 103 cmauggumagcccuugggcucTT 104 1803-1825AL-DP-6863 agcccaagggcuaccaugcTT 105 gcaugguagcccuugggcuTT 106 1803-1825AL-DP-6276 agcmcmcmaagggcmumacmcmaumgcmTT 107 gcmauggumagcccuugggcuTT108 1926-1948 AL-DP-6864 cgugcugcgugccgcaggcTT 109 gccugcggcacgcagcacgTT110 1926-1948 AL-DP-6277 cmgumgcmumgcmgumgcmcmgcmaggcmTT 111gccugcggcmacgcmagcmacgTT 112 ¹a, g, c, u: ribonucleotide5′-monophosphates (except where in 5′-most position, see below); cm:2′-O-methyl-cytidine-5′-monophosphate (except where in 5′-most position,see below); um: 2′-O-methyl-uridine-5′-monophosphate (except where in5′-most position, see below); T: deoxythymidine 5′-monophosphate; T:deoxythymidine 5′-monothiophosphate; all sequences given 5′ → 3′; due tosynthesis procedures, the 5′-most nucleic acid is anucleoside, i.e. doesnot bear a 5′-monophosate group

The iRNA agents of the invention can either contain only naturallyoccurring ribonucleotide subunits, or can be synthesized so as tocontain one or more modifications to the base, the sugar or thephosphate group of one or more of the ribonucleotide 5′-monophosphatesubunits that is included in the agent. The iRNA agent can be furthermodified so as to be attached to a ligand that is selected to improvestability, distribution or cellular uptake of the agent, e.g.cholesterol. The iRNA agents can further be in isolated form or can bepart of a pharmaceutical composition used for the methods describedherein, particularly as a pharmaceutical composition formulated fordelivery to the lung or formulated for parenteral administration. Thepharmaceutical compositions can contain one or more iRNA agents, and insome embodiments, will contain two or more iRNA agents, each onedirected to a different segment of a TGF-beta gene or a differentTGF-beta gene.

In a preferred embodiment, an iRNA agent of the invention reduces thelevel of TGF-beta protein secreted by, and/or TGF-beta mRNA foundinside, a cell by at least 20% or more. Thus, the iRNA agent maypreferably be chosen from the group of AD-14501, AD-14503, AD-14507,AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858, AL-DP-6857,AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420, AD-14421,AD-14422, AD-14423, AD-14428, AD-14430, AD-14434, AD-14438, AD-14449,AD-14453, AD-14459, AD-14460, AD-14464, AD-14469, AD-14470, AD-14473,AD-14474, AD-14476, AD-14486, AD-14490, AD-14495, AD-14496, AD-14497,AD-14509, AD-14515, AD-14522, AD-14526, AD-14540, AD-14552, AD-14555,AD-14557, AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604, AD-14608,AD-14610, AD-14612, AD-14614, AD-14622, AD-14634, AD-14635, AL-DP-6845,AL-DP-6852, AL-DP-6838, AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842,AL-DP-6837, AD-14425, AD-14426, AD-14427, AD-14429, AD-14431, AD-14436,AD-14437, AD-14441, AD-14448, AD-14458, AD-14463, AD-14467, AD-14477,AD-14482, AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548, AD-14549,AD-14559, AD-14563, AD-14564, AD-14569, AD-14578, AD-14579, AD-14587,AD-14595, AD-14599, AD-14607, AD-14609, AD-14629, AD-14631, AD-14637,AL-DP-6840, AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272, AL-DP-6271,AL-DP-6847, AL-DP-6863, AD-14447, AD-14451, AD-14471, AD-14484,AD-14487, AD-14498, AD-14523, AD-14527, AD-14529, AD-14539, AD-14541,AD-14558, AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615,AD-14626, AD-14630, AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864,AL-DP-6844, AL-DP-6143, AD-14432, AD-14480, AD-14485, AD-14488,AD-14499, AD-14504, AD-14516, AD-14518, AD-14520, AD-14547, AD-14572,AD-14577, AD-14580, AD-14583, AD-14584, AD-14618, AD-14621, AD-14625,AL-DP-6276, AD-14435, AD-14445, AD-14450, AD-14456, AD-14466, AD-14475,AD-14489, AD-14492, AD-14511, AD-14535, AD-14536, AD-14538, AD-14553,AD-14560, AD-14562, AD-14576, AD-14581, AD-14586, AD-14627, AL-DP-6150AL-DP-6850, AD-14433, AD-14472, AD-14478, AD-14493, AD-14510, AD-14544,AD-14566, AD-14570, AD-14575, AD-14593, AD-14613, AL-DP-6269,AL-DP-6270, AL-DP-6147, AL-DP-6262, AL-DP-6841, AL-DP-6274, AL-DP-6843,AL-DP-6145, AD-14424, AD-14442, AD-14444, AD-14446, AD-14454, AD-14525,AD-14543, AD-14571, AD-14606, AD-14611, AD-14616, AD-14617, AD-14619,and AD-14638 (the “20% group”).

More preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 30% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, AD-14633, AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848,AL-DP-6862, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459, AD-14460,AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515, AD-14522,AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565, AD-14567,AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598, AD-14600,AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,AD-14622, AD-14634, AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838,AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425,AD-14426, AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482, AD-14483,AD-14491, AD-14502, AD-14505, AD-14508, AD-14512, AD-14513, AD-14519,AD-14524, AD-14537, AD-14546, AD-14548, AD-14549, AD-14559, AD-14563,AD-14564, AD-14569, AD-14578, AD-14579, AD-14587, AD-14595, AD-14599,AD-14607, AD-14609, AD-14629, AD-14631, AD-14637, AL-DP-6840,AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847,AL-DP-6863, AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558, AD-14561,AD-14573, AD-14589, AD-14596, AD-14605, AD-14615, AD-14626, AD-14630,AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864, AL-DP-6844, AL-DP-6143,AD-14432, AD-14480, AD-14485, AD-14488, AD-14499, AD-14504, AD-14516,AD-14518, AD-14520, AD-14547, AD-14572, AD-14577, AD-14580, AD-14583,AD-14584, AD-14618, AD-14621, AD-14625, AL-DP-6276, AD-14435, AD-14445,AD-14450, AD-14456, AD-14466, AD-14475, AD-14489, AD-14492, AD-14511,AD-14535, AD-14536, AD-14538, AD-14553, AD-14560, AD-14562, AD-14576,AD-14581, AD-14586, AD-14627, AL-DP-6150 AL-DP-6850, AD-14433, AD-14472,AD-14478, AD-14493, AD-14510, AD-14544, AD-14566, AD-14570, AD-14575,AD-14593, and AD-14613 (the “30% group”).

More preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 40% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, AD-14633, AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848,AL-DP-6862, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459, AD-14460,AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515, AD-14522,AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565, AD-14567,AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598, AD-14600,AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,AD-14622, AD-14634, AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838,AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425,AD-14426, AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482, AD-14483,AD-14491, AD-14502, AD-14505, AD-14508, AD-14512, AD-14513, AD-14519,AD-14524, AD-14537, AD-14546, AD-14548, AD-14549, AD-14559, AD-14563,AD-14564, AD-14569, AD-14578, AD-14579, AD-14587, AD-14595, AD-14599,AD-14607, AD-14609, AD-14629, AD-14631, AD-14637, AL-DP-6840,AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847,AL-DP-6863, AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558, AD-14561,AD-14573, AD-14589, AD-14596, AD-14605, AD-14615, AD-14626, AD-14630,AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864, AL-DP-6844, AL-DP-6143,AD-14432, AD-14480, AD-14485, AD-14488, AD-14499, AD-14504, AD-14516,AD-14518, AD-14520, AD-14547, AD-14572, AD-14577, AD-14580, AD-14583,AD-14584, AD-14618, AD-14621, AD-14625, AL-DP-6276, AD-14435, AD-14445,AD-14450, AD-14456, AD-14466, AD-14475, AD-14489, AD-14492, AD-14511,AD-14535, AD-14536, AD-14538, AD-14553, AD-14560, AD-14562, AD-14576,AD-14581, AD-14586, and AD-14627 (the “40% group”).

More preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 50% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, AD-14633, AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848,AL-DP-6862, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459, AD-14460,AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515, AD-14522,AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565, AD-14567,AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598, AD-14600,AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,AD-14622, AD-14634, AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838,AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425,AD-14426, AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482, AD-14483,AD-14491, AD-14502, AD-14505, AD-14508, AD-14512, AD-14513, AD-14519,AD-14524, AD-14537, AD-14546, AD-14548, AD-14549, AD-14559, AD-14563,AD-14564, AD-14569, AD-14578, AD-14579, AD-14587, AD-14595, AD-14599,AD-14607, AD-14609, AD-14629, AD-14631, AD-14637, AL-DP-6840,AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847,AL-DP-6863, AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558, AD-14561,AD-14573, AD-14589, AD-14596, AD-14605, AD-14615, AD-14626, AD-14630,AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864, AL-DP-6844, AL-DP-6143,AD-14432, AD-14480, AD-14485, AD-14488, AD-14499, AD-14504, AD-14516,AD-14518, AD-14520, AD-14547, AD-14572, AD-14577, AD-14580, AD-14583,AD-14584, AD-14618, AD-14621, and AD-14625 (the “50% group”).

Yet more preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 60% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, AD-14633, AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848,AL-DP-6862, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459, AD-14460,AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515, AD-14522,AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565, AD-14567,AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598, AD-14600,AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,AD-14622, AD-14634, AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838,AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425,AD-14426, AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482, AD-14483,AD-14491, AD-14502, AD-14505, AD-14508, AD-14512, AD-14513, AD-14519,AD-14524, AD-14537, AD-14546, AD-14548, AD-14549, AD-14559, AD-14563,AD-14564, AD-14569, AD-14578, AD-14579, AD-14587, AD-14595, AD-14599,AD-14607, AD-14609, AD-14629, AD-14631, AD-14637, AL-DP-6840,AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847,AL-DP-6863, AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,AD-14523, AD-1.4527, AD-14529, AD-14539, AD-14541, AD-14558, AD-14561,AD-14573, AD-14589, AD-14596, AD-14605, AD-14615, AD-14626, and AD-14630(the “60% group”).

Yet more preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 70% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, AD-14633, AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848,AL-DP-6862, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459, AD-14460,AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515, AD-14522,AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565, AD-14567,AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598, AD-14600,AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,AD-14622, AD-14634, AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838,AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425,AD-14426, AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482, AD-14483,AD-14491, AD-14502, AD-14505, AD-14508, AD-14512, AD-14513, AD-14519,AD-14524, AD-14537, AD-14546, AD-14548, AD-14549, AD-14559, AD-14563,AD-14564, AD-14569, AD-14578, AD-14579, AD-14587, AD-14595, AD-14599,AD-14607, AD-14609, AD-14629, AD-14631, and AD-14637 (the “70% group”).

Yet more preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 80% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, AD-14633, AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848,AL-DP-6862, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459, AD-14460,AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515, AD-14522,AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565, AD-14567,AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598, AD-14600,AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,AD-14622, AD-14634, and AD-14635 (the “80% group”).

Yet more preferably, an iRNA agent of the invention reduces the level ofTGF-beta protein secreted by, and/or TGF-beta mRNA found inside, a cellby at least 90% or more. Thus, the iRNA agent may preferably be chosenfrom the group of AD-14501, AD-14503, AD-14507, AD-14554, AD-14594,AD-14597, and AD-14633 (the “90% group”).

The present invention provides methods for reducing the level ofTGF-beta protein and TGF-beta mRNA in a cell. Such methods optionallycomprise the step of administering one of the iRNA agents of the presentinvention to a subject as further described below. The present methodsutilize the cellular mechanisms involved in RNA interference toselectively degrade the TGF-beta mRNA in a cell and are comprised of thestep of contacting a cell with one of the TGF-beta iRNA agents of thepresent invention. Such methods can be performed directly on a cell orcan be performed on a mammalian subject by administering to a subjectone of the iRNA agents/pharmaceutical compositions of the presentinvention. Reduction of TGF-beta mRNA in a cell results in a reductionin the amount of TGF-beta protein produced, and in an organism (as shownin the Examples).

The methods and compositions of the invention, e.g., the methods andiRNA agent compositions can be used with any dosage and/or formulationdescribed herein, as well as with any route of administration describedherein.

In another aspect, the invention features a method for treating orpreventing a disease or condition associated with undesired TGF-betasignaling in a subject. The method can include administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition associated withundesired TGF-beta signaling in the subject, alone or in conjunctionwith one or more other therapeutic compounds.

In one embodiment, the iRNA agent is administered at or near the site ofundesired TGF-beta signaling, e.g., direct injection at the site, or bya catheter or other placement device (e.g., an implant including aporous, non-porous, or gelatinous material). In another embodiment theiRNA agent is administered via pulmonary delivery, e.g. by inhalation,which may be nasal and/or oral.

In one embodiment, an iRNA agent is administered repeatedly.Administration of an iRNA agent can be carried out over a range of timeperiods. It can be administered hourly, daily, once every few days,weekly, or monthly. The timing of administration can vary from patientto patient, depending upon such factors as the severity of a patient'ssymptoms. For example, an effective dose of an iRNA agent can beadministered to a patient once a month for an indefinite period of time,or until the patient no longer requires therapy. In addition, sustainedrelease compositions containing an iRNA agent can be used to maintain arelatively constant dosage in the area of the target TGF-beta nucleotidesequences.

In another embodiment, an iRNA agent is delivered at a dosage on theorder of about 0.00001 mg to about 3 mg per kg body weight of thesubject to be treated, or preferably about 0.0001-0.001 mg per kg bodyweight, about 0.03-3.0 mg per kg body weight, about 0.1-3.0 mg per kgbody weight or about 0.3-3.0 mg per kg body weight.

In another embodiment, an iRNA agent is administered prophylacticallysuch as to prevent or slow the onset of a disease or disorder orcondition associated with undesired TGF-beta signaling. For example, aniRNA can be administered to a patient who is susceptible to or otherwiseat risk for such a disease or disorder.

In another aspect, a method of inhibiting TGF-beta expression isprovided. One such method includes administering an effective amount ofan iRNA agent including sense and antisense sequences capable of formingan RNA duplex. The sense sequence of the iRNA agent can include anucleotide sequence substantially identical to a target sequence ofabout 19 to 23 nucleotides of TGF-beta mRNA, and the antisense sequencecan include a nucleotide sequence complementary to a target sequence ofabout 19-23 nucleotides of TGF-beta mRNA.

In one aspect, methods of treating any disease or disorder associatedwith undesired TGF-beta signaling are provided.

In another aspect, a method of treating idiopathic pulmonary fibrosis isprovided. One such method includes administering a therapeuticallyeffective amount of an iRNA agent that includes sense and antisensesequences capable of forming an RNA duplex. The sense sequence caninclude a nucleotide sequence substantially identical to a targetsequence of about 19 to 23 nucleotides of TGF-beta mRNA. The antisensesequence can include a nucleotide sequence complementary to a targetsequence of about 19 to 23 nucleotides of TGF-beta mRNA.

In one embodiment, a human has been diagnosed with a disease or disorderassociated with undesired TGF-beta signaling. In one embodiment, saiddisease or disorder is Interstitial Lung Disease (ILD). In a preferredembodiment, the human has been diagnosed with one of: Idiopathicpulmonary fibrosis, Hypersensitivity pneumonitis, Sarcoidosis,Eosinophilic granuloma, Wegener's granulomatosis, Idiopathic pulmonaryhemosiderosis, and Bronchiolitis obliterans. In a particularly preferredembodiment, the human has been diagnosed with idiopathic pulmonaryfibrosis.

In another aspect, the invention features a kit containing an iRNA agentof the invention. The iRNA agent of the kit can be chemically modifiedand can be useful for modulating the expression of a TGF-beta targetgene in a cell, tissue or organism. In one embodiment, the kit containsmore than one iRNA agent of the invention.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from this description and from the claims.This application incorporates all cited references, patents, and patentapplications by references in their entirety for all purposes.

DETAILED DESCRIPTION

For ease of exposition the term “nucleotide” or “ribonucleotide” issometimes used herein in reference to one or more monomeric subunits ofan RNA agent. It will be understood that the usage of the term“ribonucleotide” or “nucleotide” herein can, in the case of a modifiedRNA or nucleotide surrogate, also refer to a modified nucleotide, orsurrogate replacement moiety, as further described below, at one or morepositions.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are described herein or are wellknown in the RNA synthetic art. While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Examples include those that have a 2′ sugar modification, amodification in a single strand overhang, such as a 3′ single strandoverhang, or, particularly if single stranded, a 5′-modification whichincludes one or more phosphate groups or one or more analogs of aphosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can down-regulate the expression of atarget gene, e.g., a TGF-beta gene, and preferably a human TGF-betagene. While not wishing to be bound by theory, an iRNA agent may act byone or more of a number of mechanisms, including post-transcriptionalcleavage of a target mRNA sometimes referred to in the art as RNAinterference or RNAi, or pre-transcriptional or pre-translationalmechanisms. An iRNA agent can be a double stranded (ds) iRNA agent.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure. A “strand” herein refers to a contiguoussequence of nucleotides (including non-naturally occurring or modifiednucleotides). The two or more strands may be, or each form a part of,separate molecules, or they may be covalently interconnected, e.g. by alinker, e.g. a polyethylene glycol linker, to form but one molecule. Atleast one strand can include a region which is sufficientlycomplementary to a target RNA. Such strand is termed the “antisensestrand”. A second strand comprised in the dsRNA agent that comprises aregion complementary to the antisense strand is termed the “sensestrand”. However, a ds iRNA agent can also be formed from a single RNAmolecule which is, at least partly; self-complementary, forming, e.g., ahairpin or panhandle structure, including a duplex region. In such case,the term “strand” refers to one of the regions of the RNA molecule thatis complementary to another region of the same RNA molecule.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and/or host. The iRNA agents of thepresent invention include molecules which are sufficiently short thatthey do not trigger a deleterious interferon response in mammaliancells. Thus, the administration of a composition of an iRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of an TGF-beta gene while circumventing a deleteriousinterferon response. Molecules that are short enough that they do nottrigger a deleterious interferon response are termed siRNA agents orsiRNAs herein. “siRNA agent” or “siRNA” as used herein, refers to aniRNA agent, e.g., a ds iRNA agent, that is sufficiently short that itdoes not induce a deleterious interferon response in a human cell, e.g.,it has a duplexed region of less than 60 but preferably less than 50,40, or 30 nucleotide pairs.

The isolated iRNA agents described herein, including ds iRNA agents andsiRNA agents, can mediate silencing of a gene, e.g., by RNA degradation.For convenience, such RNA is also referred to herein as the RNA to besilenced, or target RNA. Such a gene is also referred to as a targetgene. Preferably, the RNA to be silenced is a gene product of a TGF-betagene.

As used herein, the phrases “mediate RNAi”, “silence a TGF-beta gene”,“reducing the level of TGF-beta mRNA” and/or “reducing the level ofTGF-beta protein” all refer to the at least partial suppression of theexpression of the TGF-beta gene, as manifested, e.g., by a reduction ofthe amount of mRNA transcribed from the TGF-beta gene which may beisolated from a first cell or group of cells in which the TGF-beta geneis transcribed and which has or have been treated such that theexpression of the TGF-beta gene is inhibited, as compared to a secondcell or group of cells substantially identical to the first cell orgroup of cells but which has or have not been so treated (controlcells). Alternatively, the degree of inhibition may be given in terms ofa reduction of a parameter that is functionally linked to TGF-beta genetranscription, e.g. the amount of protein encoded by the TGF-beta genewhich is secreted by a cell, or the number of cells displaying a certainphenotype associated with TGF-beta signalling. The degree of inhibitionis usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

(“mRNA” may be replaced by another parameter used to assess TGF-betagene expression, where applicable)

For example, in certain instances, expression of the TGF-beta gene issuppressed by at least about 20%, 25%, 35%, or 50% by administration ofthe double-stranded oligonucleotide of the invention. In a preferredembodiment, the TGF-beta gene is suppressed by at least about 60%, 70%,or 80% by administration of the double-stranded oligonucleotide of theinvention. In a more preferred embodiment, the TGF-beta gene issuppressed by at least about 85%, 90%, or 95% by administration of thedouble-stranded oligonucleotide of the invention. In a most preferredembodiment, the TGF-beta gene is suppressed by at least about 98%, 99%or more by administration of the double-stranded oligonucleotide of theinvention.

In principle, TGF-beta gene silencing may be determined in any cellexpressing the target, either constitutively or by genomic engineering,and by any appropriate assay. However, when a reference is needed inorder to determine whether a given siRNA inhibits the expression of theTGF-beta gene by a certain degree and therefore is encompassed by theinstant invention, the assay provided in the Examples below shall serveas such reference.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butpreferably not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, “essentially identical” when used referring to a firstnucleotide sequence in comparison to a second nucleotide sequence meansthat the first nucleotide sequence is identical to the second nucleotidesequence except for up to one, two or three nucleotide substitutions(e.g. adenosine replaced by uracil).

As used herein, a “subject” refers to a mammalian organism undergoingtreatment for a disorder associated with undesired TGF-beta signaling,such as undergoing treatment prophylactically or therapeutically toprevent TGF-beta production. The subject can be any mammal, such as aprimate, cow, horse, mouse, rat, dog, pig, goat. In the preferredembodiment, the subject is a human.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a TGF-beta gene, including mRNA that is a product of RNA processingof a primary transcription product.

As used herein, treating a disorder associated with undesired TGF-betasignaling refers to the amelioration of any biological or pathologicalendpoints that 1) is mediated in part by the unwanted expression or overexpression of TGF-beta in the subject and 2) whose outcome can beaffected by reducing the level of TGF-beta gene products present.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of painor an overt symptom of pain. The specific amount that is therapeuticallyeffective can be readily determined by ordinary medical practitioner,and may vary depending on factors known in the art, such as, e.g. thetype of pain, the patient's history and age, the stage of pain, and theadministration of other anti-pain agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

Design and Selection of iRNA Agents

The present invention is based on the demonstration of target genesilencing of the TGF-beta gene in vitro following administration of aniRNA agent results in reducing TGF-beta signaling.

Based on these results, the invention specifically provides an iRNAagent that can be used in reducing TGF-beta levels in a cell ororganism, particularly for use in reducing undesired TGF-beta signaling,in isolated form and as a pharmaceutical composition described below.Such agents will include a sense strand having at least 15 or morecontiguous nucleotides that are complementary to a TGF-beta gene and anantisense strand having at least 15 or more contiguous nucleotides thatare complementary to the sense strand sequence. Particularly useful areiRNA agents that comprise a nucleotide sequence from the TGF-beta geneas provided in Table 1 and Table 5.

Other candidate iRNA agents can be designed by performing, for example,a gene walk analysis of the TGF-beta gene that will serve as the iRNAtarget. Overlapping, adjacent, or closely spaced candidate agentscorresponding to all or some of the transcribed region can be generatedand tested. Each of the iRNA agents can be tested and evaluated for theability to down regulate the target gene expression (see below,“Evaluation of Candidate iRNA agents”).

Preferably, the iRNA agents of the present invention are based on andcomprise at least 15 or more contiguous nucleotides from one of the iRNAagents shown to be active in Table 1 and Table 5. In such agents, theagent can comprise the entire sequence provided in the table or cancomprise 15 or more contiguous residues along with additionalnucleotides from contiguous regions of the target gene. Alternatively,an iRNA agent of the instant invention may effect the cleavage of aTGF-beta mRNA within a sequence on the TGF-beta mRNA that is the targetsequence of one of the iRNA agents provided in Table 1 and Table 5. Thetarget sequences of the iRNA agents in Table 5 is expressly providedtherein, the target sequence of the iRNA agents provided in Table 1 isequal to the sense strand sequence of these agents, minus the5′-terminal deoxythymidines (and, where applicable, replacing2′-O-methyl ribonucleotides by ribonucleotides). The skilled person iswell aware of how an iRNA agent that cleaves a target mRNA within agiven mRNA sequence may be designed. Cleavage of a target mRNA occursbetween the two nucleotides hybrized to those in positions 10 and 11,counting 5′ to 3′, of the iRNA agent's antisense strand (Elbashir, S.,et al., EMBO J. 2001, 23:6877). Therefore, an iRNA agent may be designedto cleave at a given site by including a sequence in its antisensestrand sufficiently complementary to the target mRNA to hybridize to thetarget mRNA such that 10 nucleotides of the antisense strand arehybridized 3′ to the intended cleavage site on the mRNA, the remainderbeing hybridized to a sequence 5′ on the target mRNA.

An iRNA agent can be rationally designed based on sequence informationand desired characteristics and the information provided in Table 1 andTable 5. For example, an iRNA agent can be designed according to therelative melting temperature of the candidate duplex. Generally, theduplex should have a lower melting temperature at the 5′ end of theantisense strand than at the 3′ end of the antisense strand.

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Table 1 and Table 5, the dsRNAs ofthe invention can comprise at least one strand of a length of minimally21 nt. It can be reasonably expected that shorter dsRNAs comprising oneof the sequences of Table 1 and Table 5 minus only a few nucleotides onone or both ends may be similarly effective as compared to the dsRNAsdescribed above. Hence, dsRNAs comprising a partial sequence from one ofthe sequences of Table 1 and Table 5, and differing in their ability toinhibit the expression of a TGF-beta gene in an assay as describedherein below by not more than 5, 10, 15, 20, 25, or 30% inhibition froma dsRNA comprising the full sequence, are contemplated by the invention.

Accordingly, the present invention provides iRNA agents comprising asense strand and antisense strand each comprising a sequence of at least15, 16, 17, 18, 19, 20, 21 or 23 nucleotides which is essentiallyidentical to, as defined above, a portion of the TGF-beta gene.Exemplified iRNA agents include those that comprise 15 or morecontiguous nucleotides from one of the agents provided in Table 1 andTable 5.

The antisense strand of an iRNA agent should be equal to or at least,15, 16, 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It shouldbe equal to or less than 50, 40, or 30, nucleotides in length. Preferredranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. Exemplified iRNA agents include those that comprise 15 or morenucleotides from one of the agents in Table 1 and Table 5.

The sense strand of an iRNA agent should be equal to or at least 15, 16,17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equalto or less than 50, 40, or 30 nucleotides in length. Preferred rangesare 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.Exemplified iRNA agents include those that comprise 15 or morenucleotides from one of the agents Table 1 and Table 5.

The double stranded portion of an iRNA agent should be equal to or atleast, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50nucleotide pairs in length. It should be equal to or less than 50, 40,or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25,19 to 23, and 19 to 21 nucleotides pairs in length.

The agents provided in Table 1 and Table 5 are 21 nucleotides in lengthfor each strand. The iRNA agents contain a 19 nucleotide double strandedregion with a 2 nucleotide overhang on each of the 3′ ends of the agent.These agents can be modified as described herein to obtain equivalentagents comprising at least a portion of these sequences (15 or morecontiguous nucleotides) and or modifications to the oligonucleotidebases and linkages.

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to the TGF-beta gene, and are of sufficientlength in terms of nucleotides, that the iRNA agent, or a fragmentthereof, can mediate down regulation of the specific TGF-beta gene. Theantisense strands of the iRNA agents of the present invention arepreferably fully complementary to the mRNA sequences of TGF-beta gene.However, it is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of anTGF-beta mRNA.

Therefore, the iRNA agents of the instant invention include agentscomprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to one of the sequences of a TGF-beta genesuch as those agent provided in Table 1 and Table 5, except that notmore than 1, 2 or 3 nucleotides per strand, respectively, have beensubstituted by other nucleotides (e.g. adenosine replaced by uracil),while essentially retaining the ability to inhibit TGF-beta expressionin cultured human cells, as defined below. These agents will thereforepossess at least 15 or more nucleotides identical to one of thesequences of a TGF-beta gene but 1, 2 or 3 base mismatches with respectto either the target TGF-beta mRNA sequence or between the sense andantisense strand are introduced. Mismatches to the target TGF-beta mRNAsequence, particularly in the antisense strand, are most tolerated inthe terminal regions and if present are preferably in a terminal regionor regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′terminus, most preferably within 6, 5, 4, or 3 nucleotides of the5′-terminus of the sense strand or the 3′-terminus of the antisensestrand. The sense strand need only be sufficiently complementary withthe antisense strand to maintain the overall double stranded characterof the molecule.

It is preferred that the sense and antisense strands be chosen such thatthe iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule, such as those exemplified in Table 1 andTable 5. Thus, an iRNA agent contains sense and antisense strands,preferably paired to contain an overhang, e.g., one or two 5′ or 3′overhangs but preferably a 3′ overhang of 2-3 nucleotides. Mostembodiments will have a 3′ overhang. Preferred siRNA agents will havesingle-stranded overhangs, preferably 3′ overhangs, of 1 to 6,preferably 1 to 4, or more preferably 2 or 3 nucleotides, in length, onone or both ends of the iRNA agent. The overhangs can be the result ofone strand being longer than the other, or the result of two strands ofthe same length being staggered. 5′-ends are preferably phosphorylated.

Moreover, the present inventors have discovered that the presence ofonly one nucleotide overhang strengthens the interference activity ofthe dsRNA, without affecting its overall stability. dsRNA having onlyone overhang has proven particularly stable and effective in vivo, aswell as in a variety of cells, cell culture mediums, blood, and serum.Preferably, the single-stranded overhang is located at the 3′-terminalend of the antisense strand or, alternatively, at the 3′-terminal end ofthe sense strand. The dsRNA may also have a blunt end, preferablylocated at the 5′-end of the antisense strand. Such dsRNAs have improvedstability and inhibitory activity, thus allowing administration at lowdosages, i.e., less than 5 mg/kg body weight of the recipient per day.Preferably, the antisense strand of the dsRNA has a nucleotide overhangat the 3′-end, and the 5′-end is blunt. In another embodiment, one ormore of the nucleotides in the overhang is replaced with a nucleosidethiophosphate.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. Embodiments in which the twostrands of the siRNA agent are linked, e.g., covalently linked are alsoincluded. Hairpin, or other single strand structures which provide therequired double stranded region, and preferably a 3′ overhang are alsowithin the invention.

Evaluation of Candidate iRNA Agents

A candidate iRNA agent can be evaluated for its ability to down regulatetarget gene expression. For example, a candidate iRNA agent can beprovided, and contacted with a cell, e.g. a human cell that expressesTGF-beta. Alternatively, the cell can be transfected with a constructfrom which a target TGF-beta gene is expressed, thus preventing the needfor endogenous TGF-beta expression. The level of target gene expressionprior to and following contact with the candidate iRNA agent can becompared, e.g. on an mRNA or protein level. If it is determined that theamount of RNA or protein expressed from the target gene is lowerfollowing contact with the iRNA agent, then it can be concluded that theiRNA agent down-regulates target gene expression. The level of targetTGF-beta mRNA or TGF-beta protein in the cell or tissue can bedetermined by any method desired. For example, the level of target RNAcan be determined by Northern blot analysis, reverse transcriptioncoupled with polymerase chain reaction (RT-PCR), bDNA analysis, or RNAseprotection assay. The level of protein can be determined, for example,by Western blot analysis or immunofluorescence.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g.,its susceptibility to cleavage by an endonuclease or exonuclease, suchas when the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject.

When sites susceptible to cleavage are identified, a further iRNA agentcan be designed and/or synthesized wherein the potential cleavage siteis made resistant to cleavage, e.g. by introduction of a 2′-modificationon the site of cleavage, e.g. a 2′-O-methyl group. This further iRNAagent can be retested for stability, and this process may be iterateduntil an iRNA agent is found exhibiting the desired stability. Table 1provides a variety of sequence modifications as exemplars, which arenevertheless not to be understood as limiting.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting TGF-beta geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse or rat) as shown in the examples.For example, the iRNA agent can be administered to an animal, and theiRNA agent evaluated with respect to its biodistribution, stability, andits ability to inhibit TGF-beta, e.g. lower TGF-beta protein or geneexpression.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human, e.g.by inhalation, injection or infusion.

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as fluorescein; aradioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; orantigen particles for immunohistochemistry).

The iRNA agent can be evaluated with respect to its ability to downregulate TGF-beta gene expression. Levels of TGF-beta gene expression invivo can be measured, for example, by in situ hybridization, or by theisolation of RNA from tissue prior to and following exposure to the iRNAagent. Where the animal needs to be sacrificed in order to harvest thetissue, an untreated control animal will serve for comparison. TargetTGF-beta mRNA can be detected by any desired method, including but notlimited to RT-PCR, Northern blot, branched-DNA assay, or RNAaseprotection assay. Alternatively, or additionally, TGF-beta geneexpression can be monitored by performing Western blot analysis ontissue extracts treated with the iRNA agent.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., ds RNA agents, thatmediate RNAi to inhibit expression of a TGF-beta gene.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termed modified RNAs(apparently because these are typically the result of apost-transcriptional modification) are within the term unmodified RNA,as used herein. Modified RNA as used herein refers to a molecule inwhich one or more of the components of the nucleic acid, namely sugars,bases, and phosphate moieties, are different from that which occurs innature, preferably different from that which occurs in the human body.While they are referred to as modified “RNAs,” they will of course,because of the modification, include molecules which are not RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of each of the above are discussed herein.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. It may be desirable to modify one or both of the antisenseand sense strands of an iRNA agent. As nucleic acids are polymers ofsubunits or monomers, many of the modifications described below occur ata position which is repeated within a nucleic acid, e.g., a modificationof a base, or a phosphate moiety, or the non-linking O of a phosphatemoiety. In some cases the modification will occur at all of the subjectpositions in the nucleic acid but in many, and in fact in most, cases itwill not. By way of example, a modification may only occur at a 3′ or 5′terminal position, may only occur in a terminal region, e.g. at aposition on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of a strand. A modification may occur in a double strandregion, a single strand region, or in both. E.g., a phosphorothioatemodification at a non-linking O position may only occur at one or bothtermini, may only occur in a terminal regions, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand, or may occur in double strand and single strand regions,particularly at termini. Similarly, a modification may occur on thesense strand, antisense strand, or both. In some cases, the sense andantisense strand will have the same modifications or the same class ofmodifications, but in other cases the sense and antisense strand willhave different modifications, e.g., in some cases it may be desirable tomodify only one strand, e.g. the sense strand.

Two prime objectives for the introduction of modifications into iRNAagents is their stabilization towards degradation in biologicalenvironments and the improvement of pharmacological properties, e.g.pharmacodynamic properties, which are further discussed below. Othersuitable modifications to a sugar, base, or backbone of an iRNA agentare described in co-owned PCT Application No. PCT/US2004/01193, filedJan. 16, 2004. An iRNA agent can include a non-naturally occurring base,such as the bases described in co-owned PCT Application No.PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include anon-naturally occurring sugar, such as a non-carbohydrate cyclic carriermolecule. Exemplary features of non-naturally occurring sugars for usein iRNA agents are described in co-owned PCT Application No.PCT/US2004/11829 filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiralphosphorothioate linkage) useful for increasing nuclease resistance. Inaddition, or in the alternative, an iRNA agent can include a ribosemimic for increased nuclease resistance. Exemplary internucleotidelinkages and ribose mimics for increased nuclease resistance aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can include ligand-conjugated monomer subunits andmonomers for oligonucleotide synthesis. Exemplary monomers are describedin co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10,2004.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

An iRNA agent, e.g., an iRNA agent that targets TGF-beta, can haveenhanced resistance to nucleases.

For increased nuclease resistance and/or binding affinity to the target,an iRNA agent, e.g., the sense and/or antisense strands of the iRNAagent, can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE andaminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino). It isnoteworthy that oligonucleotides containing only the methoxyethyl group(MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilitiescomparable to those modified with the robust phosphorothioatemodification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality.

Preferred substituents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

One way to increase resistance is to identify cleavage sites and modifysuch sites to inhibit cleavage, as described in co-owned U.S.Application No. 60/559,917, filed on May 4, 2004. For example, thedinucleotides 5′-ua-3′, 5′-ca-3′,5′-ug-3′,5′-uu-3′, or 5′-cc-3′ canserve as cleavage sites. In certain embodiments, all the pyrimidines ofan iRNA agent carry a 2′-modification in either the sense strand, theantisense strand, or both strands, and the iRNA agent therefore hasenhanced resistance to endonucleases. Enhanced nuclease resistance canalso be achieved by modifying the 5′ nucleotide, resulting, for example,in at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide whereinthe uridine is a 2′-modified nucleotide; at least one5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′(5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide,wherein the 5′-uridine is a 2′-modified nucleotide; or at least one5′-cytidine-cytidine-3′ (5′-cc-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, as described in co-owned InternationalApplication No. PCT/US2005/018931, filed on May 27, 2005. The iRNA agentcan include at least 2, at least 3, at least 4 or at least 5 of suchdinucleotides. In a particularly preferred embodiment, the 5′ nucleotidein all occurrences of the sequence motifs 5′-ua-3′ and 5′-ca-3′ ineither the sense strand, the antisense strand, or both strands is amodified nucleotide. Preferably, the 5′ nucleotide in all occurrences ofthe sequence motifs 5′-ua-3′,5′-ca-3′ and 5′-ug-3′ in either the sensestrand, the antisense strand, or both strands is a modified nucleotide.More preferably, all pyrimidine nucleotides in the sense strand aremodified nucleotides, and the 5′ nucleotide in all occurrences of thesequence motifs 5′-ua-3′ and 5′-ca-3′ in the antisense strand aremodified nucleotides, or where the antisense strand does compriseneither of a 5′-ua-3′ and a 5′-ca-3′ motif, in all occurrences of thesequence motif 5′-ug-3′. Preferably, sites particularly prone tonuclease degradation are first determined, e.g. by mass spectrometry ofdegradation products, and only those sites are sequentially modifieduntil a desired level of stability is attained.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate), as further described below. The so-called “chimeric”oligonucleotides are those that contain two or more differentmodifications. Preferably, phosphate linker modifications are locatednear the 3′- and/or 5′-end of either or both strands, or at or nearsites particularly prone to degradative attack.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. An iRNA agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group,e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′exonucleolytic cleavage. While not being bound by theory, a 3′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

Similarly, 5′ conjugates can inhibit 5′-3′ exonucleolytic cleavage.While not being bound by theory, a 5′ conjugate, such as naproxen oribuprofen, may inhibit exonucleolytic cleavage by sterically blockingthe exonuclease from binding to the 5′-end of oligonucleotide. Evensmall alkyl chains, aryl groups, or heterocyclic conjugates or modifiedsugars (D-ribose, deoxyribose, glucose etc.) can block3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexediRNA agent includes a single-stranded nucleotide overhang on at leastone end. In preferred embodiments, the nucleotide overhang includes 1 to4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment,the unpaired nucleotide of the single-stranded overhang that is directlyadjacent to the terminal nucleotide pair contains a purine base, and theterminal nucleotide pair is a G-C pair, or at least two of the last fourcomplementary nucleotide pairs are G-C pairs. In further embodiments,the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in anexemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferredembodiments, the nucleotide overhang is on the 3′-end of the antisensestrand. In one embodiment, the iRNA agent includes the motif 5′-CGC-3′on the 3′-end of the antisense strand, such that a 2-nt overhang5′-GC-3′ is formed.

Thus, an iRNA agent can include modifications so as to inhibitdegradation, e.g., by nucleases, e.g., endonucleases or exonucleases,found in the body of a subject. These monomers are referred to herein asNRMs, or Nuclease Resistance promoting Monomers, the correspondingmodifications as NRM modifications. In many cases these modificationswill modulate other properties of the iRNA agent as well, e.g., theability to interact with a protein, e.g., a transport protein, e.g.,serum albumin, or a member of the RISC, or the ability of the first andsecond sequences to form a duplex with one another or to form a duplexwith another sequence, e.g., a target molecule.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent.

NRM modifications include some which can be placed only at the terminusand others which can go at any position. Some NRM modifications that caninhibit hybridization are preferably used only in terminal regions, andmore preferably not at the cleavage site or in the cleavage region of asequence which targets a subject sequence or gene, particularly on theantisense strand. They can be used anywhere in a sense strand, providedthat sufficient hybridization between the two strands of the ds iRNAagent is maintained. In some embodiments it is desirable to put the NRMat the cleavage site or in the cleavage region of a sense strand, as itcan minimize off-target silencing.

In most cases, the NRM modifications will be distributed differentlydepending on whether they are comprised on a sense or antisense strand.If on an antisense strand, modifications which interfere with or inhibitendonuclease cleavage should not be inserted in the region which issubject to RISC mediated cleavage, e.g., the cleavage site or thecleavage region (As described in Elbashir et al., 2001, Genes and Dev.15: 188, hereby incorporated by reference). Cleavage of the targetoccurs about in the middle of a 20 or 21 nt antisense strand, or about10 or 11 nucleotides upstream of the first nucleotide on the target mRNAwhich is complementary to the antisense strand. As used herein cleavagesite refers to the nucleotides on either side of the site of cleavage,on the target mRNA or on the iRNA agent strand which hybridizes to it.Cleavage region means the nucleotides within 1, 2, or 3 nucleotides ofthe cleavage site, in either direction.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

In a particular embodiment, the 5′-end of the antisense strand and the3′-end of the sense strand are chemically linked via a hexaethyleneglycol linker. In another embodiment, at least one nucleotide of thedsRNA comprises a phosphorothioate or phosphorodithioate groups. Thechemical bond at the ends of the dsRNA is preferably formed bytriple-helix bonds.

In certain embodiments, a chemical bond may be formed by means of one orseveral bonding groups, wherein such bonding groups are preferablypoly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycolchains. In other embodiments, a chemical bond may also be formed bymeans of purine analogs introduced into the double-stranded structureinstead of purines. In further embodiments, a chemical bond may beformed by azabenzene units introduced into the double-strandedstructure. In still further embodiments, a chemical bond may be formedby branched nucleotide analogs instead of nucleotides introduced intothe double-stranded structure. In certain embodiments, a chemical bondmay be induced by ultraviolet light.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents: U.S. Pat.Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for thepreparation of oligonucleotides having chiral phosphorus linkages; U.S.Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides havingmodified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No.5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modifiedoligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawnto, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

In some preferred embodiments, functionalized nucleoside sequences ofthe invention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment,ligand molecules may be conjugated to oligonucleotides at the5′-position by the use of a ligand-nucleoside phosphoramidite whereinthe ligand is linked to the 5′-hydroxy group directly or indirectly viaa linker. Such ligand-nucleoside phosphoramidites are typically used atthe end of an automated synthesis procedure to provide aligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.

In many cases, protecting groups are used during the preparation of thecompounds of the invention. As used herein, the term “protected” meansthat the indicated moiety has a protecting group appended thereon. Insome preferred embodiments of the invention, compounds contain one ormore protecting groups. A wide variety of protecting groups can beemployed in the methods of the invention. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.

Representative hydroxyl protecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-23.11). Further hydroxylprotecting groups, as well as other representative protecting groups,are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y, 1991.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Equivalents of these amino-protecting groups are also encompassed by thecompounds and methods of the invention.

Many solid supports are commercially available and one of ordinary skillin the art can readily select a solid support to be used in thesolid-phase synthesis steps. In certain embodiments, a universal supportis used. A universal support allows for preparation of oligonucleotideshaving unusual or modified nucleotides located at the 3′-terminus of theoligonucleotide. Universal Support 500 and Universal Support II areuniversal supports that are commercially available from Glen Research,22825 Davis Drive, Sterling, Va. For further details about universalsupports see Scott et al., Innovations and Perspectives in solid-phaseSynthesis, 3rd International Symposium, 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; andAzhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, ithas been reported that the oligonucleotide can be cleaved from theuniversal support under milder reaction conditions when oligonucleotideis bonded to the solid support via a syn-1,2-acetoxyphosphate groupwhich more readily undergoes basic hydrolysis. See Guzaev, A. I.;Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.

Ligand Conjugates

The properties of an iRNA agent, including its pharmacologicalproperties, can be influenced and tailored, for example, by theintroduction of ligands, e.g. tethered ligands.

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. The ligand or tethered ligand may bepresent on the ligand-conjugated monomer when the ligand-conjugatedmonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated into a “precursor” ligand-conjugatedmonomer subunit after a “precursor” ligand-conjugated monomer subunithas been incorporated into the growing strand. For example, a monomerhaving, e.g., an amino-terminated tether, e.g., TAP—(CH₂)_(n)NH₂ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer subunitinto the strand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor ligand-conjugated monomer by coupling the electrophilicgroup of the ligand with the terminal nucleophilic group of theprecursor ligand-conjugated monomer subunit tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophilic moleculeses, lipids, lectins, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid), proteins, protein bindingagents, integrin targeting molecules, polycationics, peptides,polyamines, and peptide mimics.

The ligand may be a naturally occurring or recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.Examples of polyamino acids include polyamino acid is a polylysine(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acidanhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinylether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamidecopolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers,or polyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic moieties, e.g., cationic lipid,cationic porphyrin, quaternary salt of a polyamine, or an alpha helicalpeptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a thyrotropin, melanotropin, surfactant proteinA, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin,bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGDpeptide mimetic.

Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. lowdensity lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA),or peptides, e.g., molecules having a specific affinity for a co-ligand,or antibodies e.g., an antibody, that binds to a specified cell typesuch as a cancer cell, endothelial cell, or bone cell. Ligands may alsoinclude hormones and hormone receptors. They can also includenon-peptidic species, such as cofactors, multivalent lactose,multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine,multivalent mannose, or multivalent fucose. The ligand can be, forexample, a lipopolysaccharide, an activator of p38 MAP kinase, or anactivator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., liver tissue, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient,which is taken up by a target cell, e.g., a proliferating cell. Theseare particularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include the B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells.

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as that or antennapedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Backbone Modifications

Specific examples of preferred modified oligonucleotides envisioned foruse in the ligand-conjugated oligonucleotides of the invention includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined here, oligonucleotides havingmodified backbones or internucleoside linkages include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of the invention,modified oligonucleotides that do not have a phosphorus atom in theirintersugar backbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described below. Itis not necessary for all positions in a given compound to be uniformlymodified. Conversely, more than one modifications may be incorporated ina single dsRNA compound or even in a single nucleotide thereof.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, salts and free-acid forms arealso included.

Representative United States Patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, 0, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

5′-Terminal Modifications

In preferred embodiments, iRNA agents are 5′ phosphorylated or include aphosphoryl analog at the 5′ prime terminus, e.g. 5′ prime terminus ofthe antisense strand. 5′-phosphate modifications of the antisense strandinclude those which are compatible with RISC mediated gene silencing.Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′);5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure. Other suitable 5′-phosphate modifications will be known tothe skilled person.

The sense strand can be modified in order to inactivate the sense strandand prevent formation of an active RISC, thereby potentially reducingoff-target effects. This can be accomplished by a modification whichprevents 5′-phosphorylation of the sense strand, e.g., by modificationwith a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATPrequirements and small interfering RNA structure in the RNA interferencepathway. Cell 107, 309-321.) Other modifications which preventphosphorylation can also be used, e.g., simply substituting the 5′-OH byH rather than O-Me. Alternatively, a large bulky group may be added tothe 5′-phosphate turning it into a phosphodiester linkage.

Disorders and Diseases Associated with Undesired TGF-Beta Signaling

Diseases that can be treated in accordance with the present inventioninclude, without limitation, kidney disorders associated with undesiredTGF-beta signaling and excessive fibrosis and/or sclerosis, such asglomerulonephritis (GN) of all etiologies, e.g., mesangial proliferativeGN, immune GN, and crescentic GN; diabetic nephropathy; renalinterstitial fibrosis and all causes of renal interstitial fibrosis,including hypertension; renal fibrosis resulting from complications ofdrug exposure, including cyclosporin treatment of transplant recipients,e.g. cyclosporin treatment; HIV-associated nephropathy, transplantnecropathy. The invention further includes the treatment of hepaticdiseases associated with excessive scarring and progressive sclerosis,including cirrhosis due to all etiologies, disorders of the biliarytree, and hepatic dysfunction attributable to infections such asinfection with hepatitis virus or parasites; pulmonary fibrosis andsymptoms associates with pulmonary fibrosis with consequential loss ofgas exchange or ability to efficiently move air into and out of thelungs, including adult respiratory distress syndrome (ARDS), chronicobstructive pulmonary disease (COPD); idiopathic pulmonary fibrosis(IPF), acute lung injury (ALI), or pulmonary fibrosis due to infectiousor toxic agents such as smoke, chemicals, allergens, or autoimmunediseases, such as systemic lupus erythematosus and scleroderma, chemicalcontact, or allergies. Fibroproliferative diseases targeted by theagents, pharmaceutical compositions, and treatment methods hereinfurther include cardiovascular diseases, such as congestive heartfailure, dilated cardiomyopathy, myocarditis, or vascular stenosisassociated with atherosclerosis, angioplasty treatment, or surgicalincisions or mechanical trauma. The invention also includes thetreatment of all collagen vascular disorders of a chronic or persistentnature including progressive systemic sclerosis, polymyositis,scleroderma, dermatomyositis, fascists, or Raynaud's syndrome, orarthritic conditions such as rheumatoid arthritis; eye diseasesassociated with fibroproliferative states, including proliferativevitreoretinopathy of any etiology or fibrosis associated with ocularsurgery such as treatment of glaucoma, retinal reattachment, cataractextraction, or drainage procedures of any kind; excessive orhypertrophic scar formation in the dermis occurring during wound healingresulting from trauma or surgical wounds.

While the above list is not meant in any way limiting, the treatment ofall the above disorders and diseases are contemplated by the invention.

Delivery of iRNA Agents to Tissues and Cells

Formulation

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “co-administration” refers to administering to a subject two ormore agents, and in particular two or more iRNA agents. The agents canbe contained in a single pharmaceutical composition and be administeredat the same time, or the agents can be contained in separate formulationand administered serially to a subject. So long as the two agents can bedetected in the subject at the same time, the two agents are said to beco-administered.

In the present methods, the iRNA agent can be administered to thesubject either as naked iRNA agent, in conjunction with a deliveryreagent, or as a recombinant plasmid or viral vector which expresses theiRNA agent. Preferably, the iRNA agent is administered as naked iRNA.

The iRNA agents described herein can be formulated for administration toa subject, preferably for administration locally to a site of fibrosisor sclerosis, or parenterally, e.g. via injection.

A formulated iRNA agent composition can assume a variety of states. Insome examples, the composition is at least partially crystalline,uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20,or 10% water). In other examples, the composition is amorphous, such asa wax. In another example, the iRNA agent is in an aqueous phase, e.g.,in a solution that includes water, this form being the preferred formfor administration via inhalation. The aqueous phase may contain othersolvents or excipients, e.g. ethanol.

The aqueous phase or the crystalline compositions can be incorporatedinto a delivery vehicle, e.g., a liposome (particularly for the aqueousphase), or a particle (e.g., a microparticle as can be appropriate for acrystalline composition). Generally, the iRNA agent composition isformulated in a manner that is compatible with the intended method ofadministration.

Formulations for direct injection and parenteral administration are wellknown in the art. Such formulation may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additives.For intravenous use, the total concentration of solutes should becontrolled to render the preparation isotonic.

The nucleic acid molecules of the present invention can also beco-administered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

An iRNA agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes aniRNA agent, e.g., a protein that complexes with the iRNA agent to forman iRNP. Still other agents include chelators, e.g., EDTA (e.g., toremove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., abroad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA agent preparation includes another iRNAagent, e.g., a second iRNA agent that can mediate RNAi with respect to asecond gene. Still other preparations can include at least three, five,ten, twenty, fifty, or a hundred or more different iRNA species. In someembodiments, the agents are directed to the same gene but differenttarget sequences. In another embodiment, each iRNA agent is directed toa different gene, e.g. TGF-beta.

An iRNA agent can be incorporated into pharmaceutical compositionssuitable for administration. For example, compositions can include oneor more iRNA agents and a pharmaceutically acceptable carrier.

The iRNA agents featured by the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. As used herein, “pharmaceutical formulations” includeformulations for human and veterinary use. Methods for preparingpharmaceutical compositions of the invention are within the skill in theart, for example as described in Remington's Pharmaceutical Science,18th ed., Mack Publishing Company, Easton, Pa. (1990), and The Scienceand Practice of Pharmacy, 2003, Gennaro et al., the entire disclosuresof which are herein incorporated by reference.

The present pharmaceutical formulations comprise an iRNA agent of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and thelike.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For solid compositions, conventional non-toxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more iRNA agents of the invention.

By “pharmaceutically acceptable formulation” is meant a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as PluronicP85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, Fundam. Clin. Pharmacol. 13:16, 1999); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., J. Pharm. Sci. 87:1308,1998; Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al., PNASUSA. 92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995;Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; and Tyler etal., PNAS USA 96:7053, 1999.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (WPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al., Chem. Rev.95:2601, 1995; Ishiwata et al., Chem. Phare. Bull. 43:1005, 1995).

Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 267:1275, 1995; Oku et al., Biochim.Biophys. Acta 1238:86, 1995). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 42:24864,1995; Choi et al., International PCT Publication No. WO 96/10391; Ansellet al., International PCT Publication No. WO 96/10390; Holland et al.,International PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

Alternatively, certain iRNA agents of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, Science 229:345, 1985; McGarry and Lindquist, Proc. Natl.Acad. Sci. USA 83:399, 1986; Scanlon et al., Proc. Natl. Acad. Sci. USA88:10591, 1991; Kashani-Sabet et al., Antisense Res. Dev. 2:3, 1992;Dropulic et al., J. Virol. 66:1432, 1992; Weerasinghe et al., J. Virol.65:5531, 1991; Ojwang et al., Proc. Natl. Acad. Sci. USA 89:10802, 1992;Chen et al., Nucleic Acids Res. 20:4581, 1992; Sarver et al., Science247:1222, 1990; Thompson et al., Nucleic Acids Res. 23:2259, 1995; Goodet al., Gene Therapy 4:45, 1997). Those skilled in the art realize thatany nucleic acid can be expressed in eukaryotic cells from theappropriate DNA/RNA vector. The activity of such nucleic acids can beaugmented by their release from the primary transcript by a enzymaticnucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCTWO 94/02595; Ohkawa et al., Nucleic Acids Symp. Ser. 27:156, 1992; Tairaet al., Nucleic Acids Res. 19:5125, 1991; Ventura et al., Nucleic AcidsRes. 21:3249, 1993; Chowrira et al., J. Biol. Chem. 269:25856, 1994).

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., Trends in Genetics 12:510, 1996) inserted into DNA orRNA vectors. The recombinant vectors can be DNA plasmids or viralvectors. iRNA agent-expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus, retrovirus, adenovirus,or alphavirus. In another embodiment, pol III based constructs are usedto express nucleic acid molecules of the invention (see for exampleThompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinantvectors capable of expressing the iRNA agents can be delivered asdescribed above, and persist in target cells. Alternatively, viralvectors can be used that provide for transient expression of nucleicacid molecules. Such vectors can be repeatedly administered asnecessary. Once expressed, the iRNA agent interacts with the target mRNAand generates an RNAi response. Delivery of iRNA agent-expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell(for a review see Couture et al., Trends in Genetics 12:510, 1996).

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” means that the carrier can betaken into the subject with no significant adverse toxicological effectson the subject. Acceptable carriers for therapeutic use are well knownin the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985), hereby incorporated by reference herein. The term isintended to include any and all solvents, dispersion media, coatings,preservatives, e.g. antibacterial and antifungal agents, isotonic andabsorption delaying agents, stabilizers, antioxidants, suspendingagents, dyes, flavoring agents, and the like, compatible withpharmaceutical administration. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Preservatives include, for example, without limitation, sodium benzoate,sorbic acid and esters of p-hydroxybenzoic acid.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-beta-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Treatment Methods and Delivery

Administration of the iRNA Agents A patient who has been diagnosed witha disorder characterized by undesired TGF-beta signaling can be treatedby administration of an iRNA agent described herein to block thenegative effects of TGF-beta, thereby alleviating the symptomsassociated with undesired TGF-beta signaling. For example, the iRNAagent can alleviate symptoms associated with a disease of the lung, suchas a fibrotic disorder. In other examples, the iRNA agent can beadministered to treat a patient who has a fibrotic or sclerotic diseaseof the kidney or liver; a fibroproliferative cardiovascular disease; acollagen vascular disorder; or any other fibroproliferative disorderassociated with undesired TGF-beta signaling.

A composition that includes an iRNA agent of the present invention,e.g., an iRNA agent that targets TGF-beta, can be delivered to a subjectby a variety of routes, depending upon whether local or systemictreatment is desired and upon the area to be treated. In general, thedelivery of the iRNA agents of the present invention is done to achievedelivery into the subject to the site of undesired TGF-beta signaling.The preferred means of administering the iRNA agents of the presentinvention is through either a local administration to the site offibrosis or sclerosis, such as the lung, e.g. by inhalation, orsystemically through enteral or parenteral administration.

Suitable enteral administration routes include oral delivery.

Suitable parenteral administration routes include intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection;subcutaneous injection, deposition including subcutaneous infusion (suchas by osmotic pumps), or transdermal application; intraperitoneal orintramuscular injection; intrathecal or intraventricular administration;intraocular, intraotic, intranasal, or intrapulmonary administration; ordirect application to the area at or near the site of undesired TGF-betasignaling, for example by a catheter or other placement device (e.g., apellet or implant comprising a porous, non-porous, or gelatinousmaterial). It is preferred that injections or infusions of the iRNAagent be given at or near the site of undesired TGF-beta signaling.

The anti-TGF-beta iRNA agents can be administered systemically, e.g.,orally or by intramuscular injection or by intravenous injection, inadmixture with a pharmaceutically acceptable carrier adapted for theroute of administration. Methods for the delivery of nucleic acidmolecules are described in Akhtar et al., Trends in Cell Bio. 2:139,1992; Delivery Strategies for Antisense Oligonucleotide Therapeutics,ed. Akhtar, 1995; Maurer et al., Mol. Membr. Biol., 16:129, 1999;Hofland and Huang, Handb. Exp. Pharmacol. 137:165, 1999; and Lee et al.,ACS Symp. Ser. 752:184, 2000, all of which are incorporated herein byreference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan etal., PCT WO 94/02595 further describe the general methods for deliveryof nucleic acid molecules. Nucleic acid molecules can be administered tocells by a variety of methods known to those of skill in the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins (see for example Gonzalez et al., BioconjugateChem. 10:1068, 1999), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722).

Another aspect of the invention provides for the delivery of iRNA agentsto the respiratory tract. The respiratory tract includes the upperairways, including the oropharynx and larynx, followed by the lowerairways, which include the trachea followed by bifurcations into thebronchi and bronchioli. The upper and lower airways are called theconductive airways. The terminal bronchioli then divide into respiratorybronchioli which then lead to the ultimate respiratory zone, thealveoli, or deep lung. The deep lung, or alveoli, are the primary targetof inhaled therapeutic aerosols for systemic delivery of iRNA agents.

Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably the iRNAagent, within the dispersion can reach the lung where it can, forexample, be readily absorbed through the alveolar region directly intoblood circulation. Pulmonary delivery can be effective both for systemicdelivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations; administration by inhalation may be oral and/or nasal.Delivery can be achieved with liquid nebulizers, aerosol-based inhalers,and dry powder dispersion devices. Metered-dose devices are preferred.One of the benefits of using an atomizer or inhaler is that thepotential for contamination is minimized because the devices are selfcontained. Dry powder dispersion devices, for example, deliver drugsthat may be readily formulated as dry powders. An iRNA composition maybe stably stored as lyophilized or spray-dried powders by itself or incombination with suitable powder carriers. The delivery of a compositionfor inhalation can be mediated by a dosing timing element which caninclude a timer, a dose counter, time measuring device, or a timeindicator which when incorporated into the device enables dose tracking,compliance monitoring, and/or dose triggering to a patient duringadministration of the aerosol medicament.

Examples of pharmaceutical devices for aerosol delivery include metereddose inhalers (MDIs), dry powder inhalers (DPIs), and air-jetnebulizers. Exemplary delivery systems by inhalation which can bereadily adapted for delivery of the subject iRNA agents are describedin, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCTapplications WO98/31346; WO98/10796; WO00/27359; WO01/54664;WO02/060412. Other aerosol formulations that may be used for deliveringthe iRNA agents are described in U.S. Pat. Nos. 6,294,153; 6,344,194;6,071,497, and PCT applications WO02/066078; WO02/053190; WO01/60420;WO00/66206. Further, methods for delivering iRNA agents can be adaptedfrom those used in delivering other oligonucleotides (e.g., an antisenseoligonucleotide) by inhalation, such as described in Templin et al.,Antisense Nucleic Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al.,Expert Opin Biol Ther, 2001, 1:979-83; Sandrasagra et al., AntisenseNucleic Acid Drug Dev, 2002, 12:177-81.

The delivery of the inventive agents may also involve the administrationof so called “pro-drugs”, i.e. formulations or chemical modifications ofa therapeutic substance that require some form of processing ortransport by systems innate to the subject organism to release thetherapeutic substance, preferably at the site where its action isdesired. For example, the human lungs can remove or rapidly degradehydrolytically cleavable deposited aerosols over periods ranging fromminutes to hours. In the upper airways, ciliated epithelia contribute tothe “mucociliary excalator” by which particles are swept from theairways toward the mouth. Pavia, D., “Lung Mucociliary Clearance,” inAerosols and the Lung: Clinical and Experimental Aspects, Clarke, S. W.and Pavia, D., Eds., Butterworths, London, 1984. In the deep lungs,alveolar macrophages are capable of phagocytosing particles soon aftertheir deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422(1993); and Brain, J. D., “Physiology and Pathophysiology of PulmonaryMacrophages,” in The Reticuloendothelial System, S. M. Reichard and J.Filkins, Eds., Plenum, New. York., pp. 315-327, 1985.

In preferred embodiments, particularly where systemic dosing with theiRNA agent is desired, the aerosoled iRNA agents are formulated asmicroparticles. Microparticles having a diameter of between 0.5 and tenmicrons can penetrate the lungs, passing through most of the naturalbarriers. A diameter of less than ten microns is required to bypass thethroat; a diameter of 0.5 microns or greater is required to avoid beingexhaled.

The iRNA agent of the invention can be delivered using an implant. Suchimplants can be biodegradable and/or biocompatible implants, or may benon-biodegradable implants. The implants may be permeable or impermeableto the active agent.

The iRNA agent of the invention can also be administered topically, e.g.to the skin, for example by patch or by direct application to thedermis, or by iontophoresis. Ointments, sprays, or droppable liquids canbe delivered by delivery systems known in the art such as applicators ordroppers.

The iRNA agent of the invention may be provided in sustained releasecompositions, such as those described in, for example, U.S. Pat. Nos.5,672,659 and 5,595,760. The use of immediate or sustained releasecompositions depends on the nature of the condition being treated. Ifthe condition consists of an acute or over-acute disorder, treatmentwith an immediate release form will be preferred over a prolongedrelease composition. Alternatively, for certain preventative orlong-term treatments, a sustained release composition may beappropriate.

In addition to treating pre-existing disorders or diseases associatedwith undesired TGF-beta signaling, iRNA agents of the invention can beadministered prophylactically in order to prevent or slow the onset ofthese and related disorders or diseases. In prophylactic applications,an iRNA of the invention is administered to a patient susceptible to orotherwise at risk of a particular disorder or disease associated withundesired TGF-beta signaling.

The iRNA agent of the invention can be administered in a single dose orin multiple doses. Where the administration of the iRNA agent of theinvention is by infusion, the infusion can be a single sustained dose orcan be delivered by multiple infusions. Injection of the agent directlyinto the tissue is at or near the site of undesired TGF-beta signalingis preferred. Multiple injections of the agent into the tissue at ornear the site of undesired TGF-beta signaling are also preferred.

Dosage. The dosage can be an amount effective to treat or prevent adisorder or disease associated with undesired TGF-beta signaling.

One skilled in the art can readily determine an appropriate dosageregimen for administering the iRNA agent of the invention to a givensubject. For example, the iRNA agent can be administered to the subjectonce, e.g., as a single injection or deposition at or near the site ofundesired TGF-beta signaling. Alternatively, the unit dose isadministered less frequently than once a day, e.g., less than every 2,4, 8 or 30 days. In another embodiment, the unit dose is notadministered with a frequency (e.g., not a regular frequency). BecauseiRNA agent mediated silencing can persist for several days afteradministering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen, or only upon the reoccurrence of a symptom ordisease state. Alternatively, the iRNA agent can be administered once ortwice daily to a subject for a period of from about three to abouttwenty-eight days, more preferably from about seven to about ten days.In a preferred dosage regimen, the iRNA agent is injected at or near asite of unwanted TGF-beta expression (such as near a site of fibrosis orsclerosis) once a day for seven days. Where a dosage regimen comprisesmultiple administrations, it is understood that the effective amount ofiRNA agent administered to the subject can comprise the total amount ofiRNA agent administered over the entire dosage regimen.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance doses are preferably administered nomore than once every 5, 10, or 30 days. Further, the treatment regimenmay last for a period of time which will vary depending upon the natureof the particular disease, its severity and the overall condition of thepatient. In preferred embodiments the dosage may be delivered no morethan once per day, e.g., no more than once per 24, 36, 48, or morehours, e.g., no more than once every 5 or 8 days. Following treatment,the patient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracistemal or intracapsular), orreservoir may be advisable.

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. administration to the lung.For example, inhalable formulations may require lower concentrations ofsome ingredients in order to avoid irritation or discomfort compared tooral formulations. It is sometimes desirable to dilute an oralformulation up to 10-100 times in order to provide a suitable inhalableformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity and responsiveness ofthe disease or disorder, previous treatments, the general health and/orage of the subject, and other diseases present. It will also beappreciated that the effective dosage of an iRNA agent such as an siRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays. For example, the subject can bemonitored after administering an iRNA agent composition. Based oninformation from the monitoring, an additional amount of the iRNA agentcomposition can be administered.

Moreover, depending on the above factors, the course of treatment maylast from several days to several months, or until a cure is effected ora diminution of disease state is achieved. Optimal dosing schedules canbe calculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models.

Dosage levels on the order of about 1 μg/kg to 100 mg/kg of body weightper administration are useful in the treatment of the disorders ordiseases associated with undesired TGF-beta signaling. The preferreddosage range is about 0.00001 mg to about 3 mg per kg body weight of thesubject to be treated, or preferably about 0.0001-0.001 mg per kg bodyweight, about 0.03-3.0 mg per kg body weight, about 0.1-3.0 mg per kgbody weight or about 0.3-3.0 mg per kg body weight.

An iRNA agent can be administered at a unit dose less than about 75 mgper kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20, 10, 5,2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight, and less than 200 nmol of iRNA agent (e.g., about 4.4×10¹⁶copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmol of iRNA agent per kg of bodyweight. The unit dose, for example, canbe administered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into an organ), an inhaled dose, or a topicalapplication.

Delivery of an iRNA agent directly to an organ (e.g., directly to theliver or lung) can be at a dosage on the order of about 0.00001 mg toabout 3 mg per organ, or preferably about 0.0001-0.001 mg per organ,about 0.03-3.0 mg per organ, about 0.1-3.0 mg per kg body weight orabout 0.3-3.0 mg per organ.

Where an initial dose/maintenance dose regimen is followed, themaintenance dose or doses are generally lower than the initial dose,e.g., one-half less of the initial dose. A maintenance regimen caninclude treating the subject with a dose or doses ranging from 0.01 μgto 75 mg/kg of body weight per day, e.g., 70, 60, 50, 40, 30, 20, 10, 5,2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight per day.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Selection for the Targeting of TGF-Beta by ShortInterfering siRNAs

siRNA design was carried out to identify siRNAs targeting human andmouse TGF-beta 1 genes. The siRNA in silico selection resulted in 28siRNAs satisfying our selection criteria.

Human (NM_(—)000660.3; May 4, 2005) and mouse (NM_(—)011577.1; Dec. 17,2003) mRNA sequences to TGF-beta were downloaded from NCBI resource onthe worldwide web atncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide.

To setup an environment for sequence analysis, BioEdit SequenceAlignment Editor software (Version 7.0.4.1; Hall, T. A., Nucl. Acids.Symp. Ser., 1999, 41:95-98) was downloaded from the worldwide web atmbio.ncsu.edu/BioEdit/bioedit.html and installed on a windows 2000computer.

The siRNA selection process was run as follows: ClustalW multiplealignment (Thompson J D, et al., Nucleic Acids Res. 1994, 22:4673-80)function of the software was used to generate a global alignment ofhuman and mouse mRNA sequences. Conserved regions were identified byembedded sequence analysis function of the software. For this, conservedregions were defined as sequence stretches with a minimum length of 19bases for all aligned sequences containing no internal gaps. In order tohave a reference sequence available for positions of conserved stretchesand finally siRNA target region coordination, a mapping file withpositions of consensus sequence relative to human mRNA sequence wasgenerated by the software and analyzed by a perl script.

The siRNA design web interface available at the Whitehead Institute forBiomedical Research (on the worldwide web at jura.wi.mit.edu/siRNAext)was used to identify all candidate siRNAs targeting the conservedregions as well as their predicted off-target hits to human and mousesequences. For this purpose, candidate siRNAs proposed by the tool weresubjected to the software embedded off-target analysis by running theNCBI blast algorithm against the NCBI human and mouse RefSeq database.

Blast results were downloaded and analyzed by a perl script in order toextract an off-target score for each candidate siRNA that was used forsiRNA ranking. Final goal for ranking and selection was to prefer siRNAswith high specificity. We defined an off-target score to expressoff-target potential of each siRNA based on the following assumptions:

1) high specificity can be predicted by weak identity to off-target hits

2) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) maycontribute more to off-target potential than rest of sequence (non-seedregion)

Thus, the identity score of the antisense strand calculated by blast wasconsidered as well as the positions of occurring mismatches. Theoff-target score was defined as the highest score out of all scores foran siRNA calculated for each off-target hit obtained by blast.

The off-target score was calculated as follows: identityscore−0.2*mismatches in seed region.

siRNAs were sorted according to off-target score (ascending). The top 28siRNAs according to the above criteria were selected and synthesized.

Designing siRNAs Against TGF-Beta mRNA

siRNA against TGF-beta mRNA were synthesized chemically using proceduresestablished in the art. Two different designs of siRNAs were synthesizedand tested for each base sequence obtained in the selection stepdescribed above, for a total of 56 siRNAs. The first group consisted ofunmodified ribonucleotides, except that the unpaired 3′-overhangs onboth strands consisted of 2′-deoxy thymidines. The second group furtherincluded a phosphorothioate linkage between the two unpaired 2′-deoxythymidines at the end of each strand, 2′O-methyl modifications at everypyrimidine base in the sense strand, and 2′O-methyl modifications atevery uridine occurring in a sequence context of 5′-ua-3′ and everycytidine occurring in a sequence context of 5′-ca-3′. Modifications inthe oligonucleotide strands were accomplished by using the appropriatemodified monomer phosphoramidite. The siRNA sequences of those siRNAssynthesized are listed in Table 1, together with the position of thetarget sequence for each siRNA in NM_(—)000660.3.

Example 2 TGF-Beta siRNA In Vitro Screening Protocol

Human lung epithelial A549 carcinoma cells were transfected withunstabilized and phosphorothioate/O-methyl exo-/endonuclease stabilizedsiRNAs to select a set of most active siRNAs for further development aspotential drug candidates in preclinical and clinical studies.

For transfections, A549 cells (ATCC, Manassas, USA, ATCC #: CCL-185)were seeded at 1.5×10⁴ cells/well on 96-well cell culture plates(Greiner Bio-One GmbH, Frickenhausen, Germany) in 100 μl growth medium(RPMI 1640, 10% fetal calf serum, 100 u/ml penicillin 100 μg/mlstreptomycin, 2 mM L-glutamine; Biochrom AG, Berlin, Germany).Transfection of each siRNA was performed 24 h post-seeding at 100 nM and10 nM final concentration of siRNA duplex in quadruplets usingLipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany). For each well,0.5 μl Lipofectamine 2000 were mixed with 12.5 μl Opti-MEM (Invitrogen)and incubated for 5 min at room temperature. To achieve a final siRNAconcentration of 100 nM in the incubation mixture, 2.5 μl of a 5 μMsolution of siRNA in annealing buffer per well were mixed with 10 μlOpti-MEM, per well. To achieve a final siRNA concentration of 10 nM, 2.5μl of a 0.5 μM solution of siRNA in annealing buffer were mixed with 10μl Opti-MEM, per well. The siRNA solution was combined with the dilutedLipofectamine 2000 solution, gently mixed and incubated for 20 minutesat room temperature to allow complex formation. In the meantime, growthmedium was removed from cells and replaced by 100 μl/well of freshmedium. 25 μl of the siRNA-Lipofectamine2000-complex solution wereapplied to the cells to a final volume of 125 μl per well and cells wereincubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator(Heraeus GmbH, Hanau).

Following incubation, TGF-beta mRNA levels were determined by QuantiGenebDNA-kit (Genospectra, Fremont, USA). The incubation mixture wasreplaced by 100 μl fresh growth medium and cells were lysed by applying50 μl of lysis mixture from the QuantiGene bDNA-kit and incubation for30 min at 53° C. Cell lysates were incubated in parallel with bDNAprobes specific to hGAPDH (10 μl lysate) and hTGF-beta (40 μl lysate) onQuantiGene Capture Plates and processed according to the manufacturer'sprotocol for the QuantiGene bDNA kit. The nucleotide sequences of thebDNA probes used for hTGF-beta and hGAPDH are given in Table 2 and Table3 below, respectively. Finally, chemoluminescence was measured in aVictor2-Light (Perkin Elmer, Wiesbaden, Germany) as relative light units(RLU) and values obtained with hGAPDH and hTGF-beta probesets werenormalized to the respective GAPDH values for each well. TGF-beta/GAPDHratios obtained with siRNAs directed against TGF-beta finally wererelated to the value obtained with the same concentration (100 nM or 10nM) of an unspecific siRNA (directed against ApoB) which was set to100%.

Effective siRNAs from the screen were further characterized by doseresponse curves (DRC). Transfections for dose response curves wereperformed at the following concentrations: 100 nM, 25 nM, 6.3 nM, 1.6nM, 0.4 nM, 0.1 nM, 25 pM according to the above protocol. The %TGF-beta mRNA knockdown again was related to a 100% value obtained froma control siRNA transfection. The dsRNA concentrations at which 50% oftheir maximal activity was achieved (Inhibitory concentration 50%, IC50)were calculated by curve fitting with the computer software Xlfit, (IDBusiness Solutions Ltd., Guildford, Surrey, UK) using the followingparameters: Dose Response One Site, 4 Parameter Logistic Model,fit=(A+((B−A)/(1+(((10^C)/x)^D)))),inv=((10^C)/((((B−A)/(y−A))−1)^(1/D))), res=(y−fit).

bDNA Probes Used in bDNA Assay for TGF-β (Generated with BayerQuantiGene Probeset Software):

TABLE 2 TGF-β-specific probes detecting hsTGF-β1 (Genbank accession no.NM_000660.3) FPL Func- SEQ Name tion Sequence ID NO hsTGFb 001 CEGGGCTCCGGTTCTGCACTCTTTTT 113 TCTCTTGGAAAGAAAGT hsTGFb 002 CECGCGGGTGACCTCCTTGGTTTTTC 114 TCTTGGAAAGAAAGT hsTGFb 003 CEGCAGCTCTGCCCGGGAGATTTTTC 115 TCTTGGAAAGAAAGT hsTGFb 004 CETTTTAACTTGAGCCTCAGCAGACT 116 TTTTCTCTTGGAAAGAAAGT hsTGFb 005 CEGGTTGCTGAGGTATCGCCAGTTTT 117 TCTCTTGGAAAGAAAGT hsTGFb 006 CEGCTGGGTGCCAGCAGCCTTTTTCT 118 CTTGGAAAGAAAGT hsTGFb 007 LECGTAGTAGTCGGCCTCAGGCTCTT 119 TTTAGGCATAGGACCCGTGTCT hsTGFb 008 LETGTGGGTTTCCACCATTAGCATTT 120 TTAGGCATAGGACCCGTGTCT hsTGFb 009 LEGCTTCTCGGAGCTCTGATGTGTTT 121 TTTAGGCATAGGACCCGTGTCT hsTGFb 010 LEGCAACACGGGTTCAGGTACCTTTT 122 TAGGCATAGGACCCGTGTCT hsTGFb 011 LEGAATTGTTGCTGTATTTCTGGTAC 123 ATTTTTAGGCATAGGACCCGTGTC T hsTGFb 012 BLTGCTTGAACTTGTCATAGATTTCG 124 T hsTGFb 013 BL TGAAGAACATATATATGCTGTGTG125 TACTC hsTGFb 014 BL GCTCCACGTGCTGCTCCAC 126

TABLE 3 GAPDH-specific probes detecting hsGAPDH (Genbank accession no.NM_002046.2): FPL Func- SEQ Name tion Sequence ID NO hGAP001 CEGAATTTGCCATGGGTGGAATTTTTTCT 127 CTTGGAAAGAAAGT hGAP002 CEGGAGGGATCTCGCTCCTGGATTTTTCT 128 CTTGGAAAGAAAGT hGAP003 CECCCCAGCCTTCTCCATGGTTTTTTCTC 129 TTGGAAAGAAAGT hGAP004 CEGCTCCCCCCTGCAAATGAGTTTTTCTC 130 TTGGAAAGAAAGT hGAP005 LEAGCCTTGACGGTGCCATGTTTTTAGGC 131 ATAGGACCCGTGTCT hGAP006 LEGATGACAAGCTTCCCGTTCTCTTTTTA 132 GGCATAGGACCCGTGTCT hGAP007 LEAGATGGTGATGGGATTTCCATTTTTTT 133 AGGCATAGGACCCGTGTCT hGAP008 LEGCATCGCCCCACTTGATTTTTTTTTAG 134 GCATAGGACCCGTGTCT hGAP009 LECACGACGTACTCAGCGCCATTTTTAGG 135 CATAGGACCCGTGTCT hGAP010 LEGGCAGAGATGATGACCCTTTTGTTTTT 136 AGGCATAGGACCCGTGTCT hGAP011 BLGGTGAAGACGCCAGTGGACTC 137

Table 4 shows the relative activity of the dsRNA duplexes of Table 1towards reducing TGF-beta mRNA in A549 cells after incubation with therespective dsRNA at 100 nM or 10 nM concentration, expressed as % of theTGF-beta mRNA concentration found in A549 cells that were similarlytreated except for incubation with the same concentration of a dsRNAspecific for ApoB. In addition, IC50 are given for select duplexes.

TABLE 4 Activity of siRNAs in lowering TGF-beta mRNA in A549 cellscompared to A549 cells treated with a control siRNA specific for ApoBremaining TGF-beta RNA in % of control at 100 nM at 10 nM Duplexidentifier duplex conc. duplex conc. IC(50) [nM] AL-DP-6837 29 ± 5%  47± 19% AL-DP-6140 84 ± 7%  88 ± 9%  AL-DP-6838 22 ± 12% 34 ± 8%  0.67AL-DP-6141 81 ± 16% 118 ± 13%  AL-DP-6839 99 ± 10% 105 ± 5%  AL-DP-614287 ± 3%  115 ± 18%  AL-DP-6840 31 ± 6%  37 ± 1%  AL-DP-6143 48 ± 3%  86± 7%  23 AL-DP-6841 75 ± 13% 65 ± 29% AL-DP-6144 80 ± 7%  94 ± 12%AL-DP-6842 28 ± 10% 27 ± 4%  0.12 AL-DP-6145 78 ± 9%  100 ± 22% AL-DP-6843 77 ± 9%  69 ± 13% AL-DP-6146 81 ± 3%  102 ± 4%  AL-DP-6844 48± 7%  45 ± 20% AL-DP-6147 71 ± 18% 99 ± 12% AL-DP-6845 20 ± 17% 29 ± 5% 0.16 AL-DP-6148 87 ± 20% 98 ± 11% AL-DP-6846 40 ± 14% 49 ± 29%AL-DP-6149 90 ± 13% 96 ± 13% AL-DP-6847 36 ± 22% 46 ± 27% AL-DP-6150 66± 22% 75 ± 9%  AL-DP-6848 14 ± 5%  20 ± 6%  0.31 AL-DP-6151 95 ± 5%  122± 20%  AL-DP-6849 33 ± 12% 31 ± 9%  AL-DP-6262 75 ± 5%  113 ± 4% AL-DP-6850 68 ± 30% 74 ± 6%  AL-DP-6263 102 ± 27%  142 ± 19%  AL-DP-685126 ± 11% 52 ± 11% AL-DP-6264 23 ± 3%  59 ± 9%  4.4 AL-DP-6852 21 ± 9% 32 ± 18% AL-DP-6265 86 ± 15% 79 ± 16% AL-DP-6853 41 ± 12% 52 ± 14%AL-DP-6266 83 ± 3%  97 ± 32% AL-DP-6854 92 ± 12% 118 ± 33%  AL-DP-626779 ± 12% 83 ± 11% AL-DP-6855 22 ± 2%  28 ± 6%  AL-DP-6268 88 ± 15% 107 ±46%  AL-DP-6856 43 ± 4%  54 ± 3%  AL-DP-6269 70 ± 11% 92 ± 24%AL-DP-6857 13 ± 5%  35 ± 24% 0.04 AL-DP-6270 71 ± 15% 89 ± 30%AL-DP-6858 11 ± 3%  28 ± 17% 0.13 AL-DP-6271 34 ± 12% 51 ± 15% 0.68AL-DP-6859 14 ± 2%  26 ± 11% 0.15 AL-DP-6272 33 ± 5%  56 ± 6%  2.2AL-DP-6860 31 ± 1%  45 ± 11% AL-DP-6273 118 ± 29%  161 ± 17%  AL-DP-686132 ± 7%  65 ± 37% AL-DP-6274 76 ± 4%  111 ± 11%  AL-DP-6862 16 ± 3%  27± 14% 0.19 AL-DP-6275 93 ± 41% 96 ± 20% AL-DP-6863 36 ± 17% 37 ± 15%AL-DP-6276 56 ± 12% 74 ± 32% AL-DP-6864 46 ± 26% 62 ± 19% AL-DP-6277 110± 18%  83 ± 40%

From Table 4, it can be seen that, at 100 nM concentration, AL-DP-6858,AL-DP-6857, AL-DP-6859, AL-DP-6848, and AL-DP-6862 reduced TGF-beta mRNAin A549 cells by more than 80%. Additionally, under these circumstances,AL-DP-6845, AL-DP-6852, AL-DP-6838, AL-DP-6855, AL-DP-6264, AL-DP-6851,AL-DP-6842, and AL-DP-6837 reduced TGF-beta mRNA in A549 cells by morethan 70%. Additionally, AL-DP-6840, AL-DP-6860, AL-DP-6861, AL-DP-6849,AL-DP-6272, AL-DP-6271, AL-DP-6847, AL-DP-6863 reduced TGF-beta mRNA inA549 cells by more than 60%. Additionally, AL-DP-6846, AL-DP-6853,AL-DP-6856, AL-DP-6864, AL-DP-6844, AL-DP-6143 reduced TGF-beta mRNA inA549 cells by more than 50%. Additionally, AL-DP-6276 reduced TGF-betamRNA in A549 cells by more than 40%. Additionally, AL-DP-6150 andAL-DP-6850 reduced TGF-beta mRNA in A549 cells by more than 30%.Finally, AL-DP-6269, AL-DP-6270, AL-DP-6147, AL-DP-6262, AL-DP-6841,AL-DP-6274, AL-DP-6843, AL-DP-6145, and AL-DP-6267 reduced TGF-beta mRNAin A549 cells by more than 20%.

Example 3 Screening of Additional siRNA for Activity in InhibitingTGF-Beta Expression

In order to identify as many RNAi agents with suitable activity aspossible, the above screen was extended to further target sequences. Forthe selection of an expanded screening set we re-calculated thepredicted specificity based on the newly available human RefSeq database(Human mRNA sequences in RefSeq release version 21 (downloaded Jan. 12,2007)) and selected only those 220 non-poly-G siRNAs (no occurrence of 3or more consecutive guanosines) targeting 19 mer target sequences inNM_(—)000660.3 with off-target scores of 3 or more for the antisensestrand. These agents were then screened for their activity in loweringthe amount of TGF-beta mRNA present in A549 cells treated with theagents as described above, except that transfection with the agents wasperformed directly after seeding, rather than 24 h after seeding, andthat the concentration of the agents in the transfection mixture was 30nM.

As may be concluded from the results shown in Table 5, AD-14501,AD-14503, AD-14507, AD-14554, AD-14594, AD-14597, and AD-14633 reducedTGF-beta mRNA in A549 cells by more than 90%. Additionally, under thesecircumstances, AD-14419, AD-14420, AD-14421, AD-14422, AD-14423,AD-14428, AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459,AD-14460, AD-14464, AD-14469, AD-14470, AD-14473, AD-14474, AD-14476,AD-14486, AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557, AD-14565,AD-14567, AD-14568, AD-14582, AD-14588, AD-14590, AD-14592, AD-14598,AD-14600, AD-14601, AD-14603, AD-14604, AD-14608, AD-14610, AD-14612,AD-14614, AD-14622, AD-14634, and AD-14635 reduced TGF-beta mRNA in A549cells by more than 80%. Additionally, under these circumstances,AD-14425, AD-14426, AD-14427, AD-14429, AD-14431, AD-14436, AD-14437,AD-14441, AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512, AD-14513,AD-14519, AD-14524, AD-14537, AD-14546, AD-14548, AD-14549, AD-14559,AD-14563, AD-14564, AD-14569, AD-14578, AD-14579, AD-14587, AD-14595,AD-14599, AD-14607, AD-14609, AD-14629, AD-14631, and AD-14637 reducedTGF-beta mRNA in A549 cells by more than 70%. Additionally, under thesecircumstances, AD-14447, AD-14451, AD-14471, AD-14484, AD-14487,AD-14498, AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558,AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615, AD-14626,and AD-14630 reduced TGF-beta mRNA in A549 cells by more than 60%.Additionally, under these circumstances, AD-14432, AD-14480, AD-14485,AD-14488, AD-14499, AD-14504, AD-14516, AD-14518, AD-14520, AD-14547,AD-14572, AD-14577, AD-14580, AD-14583, AD-14584, AD-14618, AD-14621,and AD-14625 reduced TGF-beta mRNA in A549 cells by more than 50%.Additionally, under these circumstances, AD-14435, AD-14445, AD-14450,AD-14456, AD-14466, AD-14475, AD-14489, AD-14492, AD-14511, AD-14535,AD-14536, AD-14538, AD-14553, AD-14560, AD-14562, AD-14576, AD-14581,AD-14586, and AD-14627 reduced TGF-beta mRNA in A549 cells by more than40%. Additionally, under these circumstances, AD-14433, AD-14472,AD-14478, AD-14493, AD-14510, AD-14544, AD-14566, AD-14570, AD-14575,AD-14593, and AD-14613 reduced TGF-beta mRNA in A549 cells by more than30%. Additionally, under these circumstances, AD-14424, AD-14442,AD-14444, AD-14446, AD-14454, AD-14525, AD-14543, AD-14571, AD-14606,AD-14611, AD-14616, AD-14617, AD-14619, and AD-14638 reduced TGF-betamRNA in A549 cells by more than 20%.

TABLE 5 Additional RNAi agents targeting hTGF-beta Remaining TGF-betamRNA target SEQ SEQ in % of Duplex 19 mer target sequence in IDAntisense strand ID controls ± identifier sequence NM_000660.3 Sensestrand sequence NO: sequence NO: stand. dev. AD-14419aauuccuggcgauaccuca 1396-1414 aauuccuggcgauaccucaTT 138ugagguaucgccaggaauuTT 139 16 ± 1 AD-14420 ucgcgcccaucuagguuau 654-672ucgcgcccaucuagguuauTT 140 auaaccuagaugggcgcgaTT 141 19 ± 2 AD-14421ggucacccgcgugcuaaug 1188-1206 ggucacccgcgugcuaaugTT 142cauuagcacgcgggugaccTT 143 19 ± 2 AD-14422 aggucacccgcgugcuaau 1187-1205aggucacccgcgugcuaauTT 144 auuagcacgcgggugaccuTT 145 10 ± 1 AD-14423cacccgcgugcuaauggug 1191-1209 cacccgcgugcuaauggugTT 146caccauuagcacgcgggugTT 147 18 ± 1 AD-14424 uacaaccagcauaacccgg 1894-1912uacaaccagcauaacccggTT 148 ccggguuaugcugguuguaTT 149 79 ± 5 AD-14425aucgcgcccaucuagguua 653-671 aucgcgcccaucuagguuaTT 150uaaccuagaugggcgcgauTT 151 24 ± 1 AD-14426 guaccagaucgcgcccauc 646-664guaccagaucgcgcccaucTT 152 gaugggcgcgaucugguacTT 153 21 ± 1 AD-14427agacggaucucucuccgac 589-607 agacggaucucucuccgacTT 154gucggagagagauccgucuTT 155 26 ± 4 AD-14428 cgcgcccaucuagguuauu 655-673cgcgcccaucuagguuauuTT 156 aauaaccuagaugggcgcgTT 157 19 ± 1 AD-14429cccgcgcauccuagacccu 558-576 cccgcgcauccuagacccuTT 158agggucuaggaugcgcgggTT 159 24 ± 3 AD-14430 gcgcccaucuagguuauuu 656-674gcgcccaucuagguuauuuTT 160 aaauaaccuagaugggcgcTT 161 18 ± 1 AD-14431uccgugggauacugagaca 674-692 uccgugggauacugagacaTT 162ugucucaguaucccacggaTT 163 25 ± 3 AD-14432 cuuucgccuuagcgcccac 1515-1533cuuucgccuuagcgcccacTT 164 gugggcgcuaaggcgaaagTT 165 41 ± 2 AD-14433acccgcgugcuaauggugg 1192-1210 acccgcgugcuaaugguggTT 166ccaccauuagcacgcggguTT 167 60 ± 4 AD-14434 gcaacaauuccuggcgaua 1391-1409gcaacaauuccuggcgauaTT 168 uaucgccaggaauuguugcTT 169 10 ± 0 AD-14435aacggguucacuaccggcc 1576-1594 aacggguucacuaccggccTT 170ggccgguagugaacccguuTT 171 57 ± 4 AD-14436 cggguucacuaccggccgc 1578-1596cggguucacuaccggccgcTT 172 gcggccgguagugaacccgTT 173 28 ± 3 AD-14437cuguauuuaaggacacccg 2117-2135 cuguauuuaaggacacccgTT 174cggguguccuuaaauacagTT 175 21 ± 1 AD-14438 agguuauuuccgugggaua 666-684agguuauuuccgugggauaTT 176 uaucccacggaaauaaccuTT 177 19 ± 1 AD-14439cccucgggagucgccgacc 458-476 cccucgggagucgccgaccTT 178ggucggcgacucccgagggTT 179 90 ± 4 AD-14440 ugugcggcagugguugagc 1476-1494ugugcggcagugguugagcTT 180 gcucaaccacugccgcacaTT 181 375 ± 34 AD-14441caaccagcauaacccgggc 1896-1914 caaccagcauaacccgggcTT 182gcccggguuaugcugguugTT 183 28 ± 4 AD-14442 uuuugagacuuuuccguug 281-299uuuugagacuuuuccguugTT 184 caacggaaaagucucaaaaTT 185 75 ± 2 AD-14443uuugagacuuuuccguugc 282-300 uuugagacuuuuccguugcTT 186gcaacggaaaagucucaaaTT 187 98 ± 9 AD-14444 cucuuggcgcgacgcugcc 326-344cucuuggcgcgacgcugccTT 188 ggcagcgucgcgccaagagTT 189 70 ± 4 AD-14445ucugguaccagaucgcgcc 642-660 ucugguaccagaucgcgccTT 190ggcgcgaucugguaccagaTT 191 50 ± 2 AD-14446 acaccagcccuguucgcgc 817-835acaccagcccuguucgcgcTT 192 gcgcgaacagggcugguguTT 193 71 ± 4 AD-14447cggccggccgcgggacuau 940-958 cggccggccgcgggacuauTT 194auagucccgcggccggccgTT 195 38 ± 1 AD-14448 gguaccugaacccguguug 1296-1314gguaccugaacccguguugTT 196 caacacggguucagguaccTT 197 21 ± 1 AD-14449guaccugaacccguguugc 1297-1315 guaccugaacccguguugcTT 198gcaacacggguucagguacTT 199 17 ± 1 AD-14450 auuccuggcgauaccucag 1397-1415auuccuggcgauaccucagTT 200 cugagguaucgccaggaauTT 201 58 ± 6 AD-14451auugagggcuuucgccuua 1507-1525 auugagggcuuucgccuuaTT 202uaaggcgaaagcccucaauTT 203 31 ± 3 AD-14452 acaaccagcauaacccggg 1895-1913acaaccagcauaacccgggTT 204 cccggguuaugcugguuguTT 205 87 ± 8 AD-14453uguauuuaaggacacccgu 2118-2136 uguauuuaaggacacccguTT 206acggguguccuuaaauacaTT 207 16 ± 2 AD-14454 gaggacugcggaucucugu 2174-2192gaggacugcggaucucuguTT 208 acagagauccgcaguccucTT 209 77 ± 5 AD-14455gggaacacuacuguaguua 2324-2342 gggaacacuacuguaguuaTT 210uaacuacaguaguguucccTT 211 86 ± 4 AD-14456 accagcccuguucgcgcuc 819-837accagcccuguucgcgcucTT 212 gagcgcgaacagggcugguTT 213 59 ± 0 AD-14457agaggacugcggaucucug 2173-2191 agaggacugcggaucucugTT 214cagagauccgcaguccucuTT 215 91 ± 3 AD-14458 guacuacgugggccgcaag 1968-1986guacuacgugggccgcaagTT 216 cuugcggcccacguaguacTT 217 24 ± 1 AD-14499gaucgcgcccaucuagguu 652-670 gaucgcgcccaucuagguuTT 218aaccuagaugggcgcgaucTT 219 13 ± 1 AD-14460 aacgaaaucuaugacaagu 1219-1237aacgaaaucuaugacaaguTT 220 acuugucauagauuucguuTT 221 16 ± 2 AD-14461uucgccuuagcgcccacug 1517-1535 uucgccuuagcgcccacugTT 222cagugggcgcuaaggcgaaTT 223 107 ± 6  AD-14462 aucaacggguucacuaccg1573-1591 aucaacggguucacuaccgTT 224 cgguagugaacccguugauTT 225 107 ± 3 AD-14463 ccgcgcauccuagacccuu 559-577 ccgcgcauccuagacccuuTT 226aagggucuaggaugcgcggTT 227 24 ± 3 AD-14464 gcgcauccuagacccuuuc 561-579gcgcauccuagacccuuucTT 228 gaaagggucuaggaugcgcTT 229 15 ± 1 AD-14465uuucgccuuagcgcccacu 1516-1534 uuucgccuuagcgcccacuTT 230agugggcgcuaaggcgaaaTT 231 85 ± 6 AD-14466 cugguaccagaucgcgccc 643-661cugguaccagaucgcgcccTT 232 gggcgcgaucugguaccagTT 233 54 ± 3 AD-14467cagaucgcgcccaucuagg 650-668 cagaucgcgcccaucuaggTT 234ccuagaugggcgcgaucugTT 235 25 ± 1 AD-14468 uccuaccuuuugccgggag 763-781uccuaccuuuugccgggagTT 236 cucccggcaaaagguaggaTT 237 93 ± 5 AD-14469cccuguucgcgcucucggc 824-842 cccuguucgcgcucucggcTT 238gccgagagcgcgaacagggTT 235 16 ± 2 AD-14470 ccuguucgcgcucucggca 825-843ccuguucgcgcucucggcaTT 240 ugccgagagcgcgaacaggTT 241 16 ± 3 AD-14471cuguucgcgcucucggcag 826-844 cuguucgcgcucucggcagTT 242cugccgagagcgcgaacagTT 243 32 ± 3 AD-14472 aagacuaucgacauggagc 967-985aagacuaucgacauggagcTT 244 gcuccaugucgauagucuuTT 245 63 ± 9 AD-14473cccgcgugcuaauggugga 1193-1211 cccgcgugcuaaugguggaTT 246uccaccauuagcacgcgggTT 247 12 ± 2 AD-14474 cgugcuaaugguggaaacc 1157-1215cgugcuaaugguggaaaccTT 248 gguuuccaccauuagcacgTT 249 13 ± 1 AD-14475ccggaguugugcggcagug 1469-1487 ccggaguugugcggcagugTT 250cacugccgcacaacuccggTT 251 52 ± 6 AD-14476 gcuuucgccuuagcgccca 1514-1532gcuuucgccuuagcgcccaTT 252 ugggcgcuaaggcgaaagcTT 253 16 ± 0 AD-14477agggauaacacacugcaag 1549-1567 agggauaacacacugcaagTT 254cuugcaguguguuaucccuTT 255 22 ± 1 AD-14478 caucaacggguucacuacc 1572-1590caucaacggguucacuaccTT 256 gguagugaacccguugaugTT 257 68 ± 1 AD-14479uuuaaggacacccgugccc 2122-2140 uuuaaggacacccgugcccTT 258gggcacggguguccuuaaaTT 259 92 ± 5 AD-14480 gacuuuuccguugccgcug 287-305gacuuuuccguugccgcugTT 260 cagcggcaacggaaaagucTT 261 41 ± 5 AD-14481uuuuccguugccgcuggga 290-308 uuuuccguugccgcugggaTT 262ucccagcggcaacggaaaaTT 263 81 ± 3 AD-14482 gggaccucuuggcgcgacg 321-339gggaccucuuggcgcgacgTT 264 cgucgcgccaagaggucccTT 265 26 ± 1 AD-14483ggaccucuuggcgcgacgc 322-340 ggaccucuuggcgcgacgcTT 266gcgucgcgccaagagguccTT 267 28 ± 3 AD-14484 gaccucuuggcgcgacgcu 323-341gaccucuuggcgcgacgcuTT 268 agcgucgcgccaagaggucTT 269 33 ± 1 AD-14485ccuacacggcgucccucag 419-437 ccuacacggcgucccucagTT 270cugagggacgccguguaggTT 271 49 ± 1 AD-14486 cgcgcauccuagacccuuu 560-578cgcgcauccuagacccuuuTT 272 aaagggucuaggaugcgcgTT 273 15 ± 2 AD-14487cauccuagacccuuucucc 564-582 cauccuagacccuuucuccTT 274ggagaaagggucuaggaugTT 275 33 ± 7 AD-14488 ugguaccagaucgcgccca 644-662ugguaccagaucgcgcccaTT 276 ugggcgcgaucugguaccaTT 277 49 ± 5 AD-14489uaccagaucgcgcccaucu 647-665 uaccagaucgcgcccaucuTT 278agaugggcgcgaucugguaTT 279 59 ± 6 AD-14490 agaucgcgcccaucuaggu 651-669agaucgcgcccaucuagguTT 280 accuagaugggcgcgaucuTT 281 19 ± 3 AD-14491gcccaucuagguuauuucc 658-676 gcccaucuagguuauuuccTT 282ggaaauaaccuagaugggcTT 283 25 ± 1 AD-14492 cuaccuuuugccgggagac 765-783cuaccuuuugccgggagacTT 284 gucucccggcaaaagguagTT 285 55 ± 2 AD-14493guucgcgcucucggcagug 828-846 guucgcgcucucggcagugTT 286cacugccgagagcgcgaacTT 287 66 ± 9 AD-14494 uucgcgcucucggcagugc 829-847uucgcgcucucggcagugcTT 288 gcacugccgagagcgcgaaTT 289 102 ± 12 AD-14495accugcaagacuaucgaca 961-979 accugcaagacuaucgacaTT 290ugucgauagucuugcagguTT 291 19 ± 1 AD-14496 cugcaagacuaucgacaug 963-981cugcaagacuaucgacaugTT 292 caugucgauagucuugcagTT 293 14 ± 1 AD-14497gcaagacuaucgacaugga 965-983 gcaagacuaucgacauggaTT 294uccaugucgauagucuugcTT 295 15 ± 1 AD-14498 caagacuaucgacauggag 966-984caagacuaucgacauggagTT 296 cuccaugucgauagucuugTT 297 37 ± 3 AD-14499uguacaacagcacccgcga 1106-1124 uguacaacagcacccgcgaTT 298ucgcgggugcuguuguacaTT 299 42 ± 2 AD-14500 ugaggccgacuacuacgcc 1164-1182ugaggccgacuacuacgccTT 300 ggcguaguagucggccucaTT 301 122 ± 7  AD-14501aaacccacaacgaaaucua 1211-1229 aaacccacaacgaaaucuaTT 302uagauuucguuguggguuuTT 303  9 ± 1 AD-14502 ccacaacgaaaucuaugac 1215-1233ccacaacgaaaucuaugacTT 304 gucauagauuucguuguggTT 305 24 ± 2 AD-14503aguacacacagcauauaua 1246-1264 aguacacacagcauauauaTT 306uauauaugcuguguguacuTT 307  8 ± 0 AD-14504 cgguaccugaacccguguu 1295-1313cgguaccugaacccguguuTT 308 aacacggguucagguaccgTT 309 45 ± 4 AD-14505uaccugaacccguguugcu 1298-1316 uaccugaacccguguugcuTT 310agcaacacggguucagguaTT 311 29 ± 2 AD-14506 cccguguugcucucccggg 1306-1324cccguguugcucucccgggTT 312 cccgggagagcaacacgggTT 313 107 ± 5  AD-14507cugcgucugcugaggcuca 1330-1348 cugcgucugcugaggcucaTT 314ugagccucagcagacgcagTT 315  9 ± 1 AD-14508 aggcucaaguuaaaagugg 1342-1360aggcucaaguuaaaaguggTT 316 ccacuuuuaacuugagccuTT 317 20 ± 1 AD-14509caacaauuccuggcgauac 1392-1410 caacaauuccuggcgauacTT 318guaucgccaggaauuguugTT 319 14 ± 1 AD-14510 aacaauuccuggcgauacc 1393-1411aacaauuccuggcgauaccTT 320 gguaucgccaggaauuguuTT 321 65 ± 3 AD-14511uggcgauaccucagcaacc 1402-1420 uggcgauaccucagcaaccTT 322gguugcugagguaucgccaTT 323 58 ± 5 AD-14512 cgauaccucagcaaccggc 1405-1423cgauaccucagcaaccggcTT 324 gccgguugcugagguaucgTT 32S 20 ± 0 AD-14513gauaccucagcaaccggcu 1406-1424 gauaccucagcaaccggcuTT 326agccgguugcugagguaucTT 327 24 ± 1 AD-14514 cuggcacccagcgacucgc 1426-1444cuggcacccagcgacucgcTT 328 gcgagucgcugggugccagTT 329  86 ± 13 AD-14515gacucgccagagugguuau 1438-1456 gacucgccagagugguuautT 330auaaccacucuggcgagucTT 331 11 ± 1 AD-14516 acucgccagagugguuauc 1439-1457acucgccagagugguuaucTT 332 gauaaccacucuggcgaguTT 333 47 ± 2 AD-14517ugucaccggaguugugcgg 1464-1482 ugucaccggaguugugcggTT 334ccgcacaacuccggugacaTT 335 115 ± 7  AD-14518 caccggaguugugcggcag1467-1485 caccggaguugugcggcagTT 336 cugccgcacaacuccggugTT 337 44 ± 3AD-14519 accggaguugugcggcagu 1468-1486 accggaguugugcggcaguTT 338acugccgcacaacuccgguTT 339 28 ± 2 AD-14520 aaauugagggcuuucgccu 1505-1523aaauugagggcuuucgccuTT 340 aggcgaaagcccucaauuuTT 341 46 ± 2 AD-14521uugagggcuuucgccuuag 1508-1526 uugagggcuuucgccuuagTT 342cuaaggcgaaagcccucaaTT 343 130 ± 15 AD-14522 gggcuuucgccuuagcgcc1512-1530 gggcuuucgccuuagcgccTT 344 ggcgcuaaggcgaaagcccTT 345 14 ± 1AD-14523 ccuuagcgcccacugcucc 1521-1539 ccuuagcgcccacugcuccTT 346ggagcagugggcgcuaaggTT 347 30 ± 1 AD-14524 aaguggacaucaacggguu 1565-1583aaguggacaucaacggguuTT 348 aacccguugauguccacuuTT 349 20 ± 1 AD-14525caacggguucacuaccggc 1575-1593 caacggguucacuaccggcTT 350gccgguagugaacccguugTT 351 73 ± 6 AD-14526 cuaccggccgccgagguga 1586-1604cuaccggccgccgaggugaTT 352 ucaccucggcggccgguagTT 353 18 ± 1 AD-14527caucugcaaagcucccggc 1675-1693 caucugcaaagcucccggcTT 354gccgggagcuuugcagaugTT 355 37 ± 2 AD-14528 cguguacuacgugggccgc 1965-1983cguguacuacgugggccgcTT 356 gcggcccacguaguacacgTT 357 80 ± 4 AD-14529caacaugaucgugcgcucc 2007-2025 caacaugaucgugcgcuccTT 358ggagcgcacgaucauguugTT 359 35 ± 2 AD-14530 cugugucauugggcgccug 2189-2207cugugucauugggcgccugTl 360 caggcgcccaaugacacagTT 361 89 ± 6 AD-14531ugccugucugcacuauucc 2273-2291 ugccugucugcacuauuccTT 362ggaauagugcagacaggcaTT 363 97 ± 4 AD-14532 cacuauuccuuugcccggc 2283-2301cacuauuccuuugcccggcTT 364 gccgggcaaaggaauagugTT 365 98 ± 8 AD-14533aacacuacuguaguuagau 2327-2345 aacacuacuguaguuagauTT 366aucuaacuacaguaguguuTT 367 89 ± 8 AD-14534 acacuacuguaguuagauc 2328-2346acacuacuguaguuagaucTT 368 gaucuaacuacaguaguguTT 369 80 ± 2 AD-14535gucugagacgagccgccgc 185-203 gucugagacgagccgccgcTT 370gcggcggcucgucucagacTT 371 57 ± 4 AD-14536 cccuacacggcgucccuca 418-436cccuacacggcgucccucaTT 372 ugagggacgccguguagggTT 373 50 ± 4 AD-14537acggaucucucuccgaccu 591-609 acggaucucucuccgaccuTT 374aggucggagagagauccguTT 375 23 ± 3 AD-14538 ggccggccgcgggacuauc 941-959ggccggccgcgggacuaucTT 376 gauagucccgcggccggccTT 377 50 ± 2 AD-14539cgcggccagauccugucca 1015-1033 cgcggccagauccuguccaTT 378uggacaggaucuggccgcgTT 379 34 ± 3 AD-14540 ggaaacccacaacgaaauc 1209-1227ggaaacccacaacgaaaucTT 380 gauuucguuguggguuuccTT 381 11 ± 0 AD-14541acaauuccuggcgauaccu 1394-1412 acaauuccuggcgauaccuTT 382agguaucgccaggaauuguTT 383 31 ± 2 AD-14542 agucugagacgagccgccg 184-202agucugagacgagccgccgTT 384 cggcggcucgucucagacuTT 385 87 ± 1 AD-14543cacggcgucccucaggcgc 423-441 cacggcgucccucaggcgcTT 386gcgccugagggacgccgugTT 387 76 ± 3 AD-14544 acggcgucccucaggcgcc 424-442acggcgucccucaggcgccTT 388 ggcgccugagggacgccguTT 389 68 ± 3 AD-14545accagcccucgggagucgc 453-471 accagcccucgggagucgcTT 390gcgacucccgagggcugguTT 391 96 ± 7 AD-14546 gcauccuagacccuuucuc 563-S81gcauccuagacccuuucucTT 392 gagaaagggucuaggaugcTT 393 22 ± 2 AD-14547gacggaucucucuccgacc 590-608 gacggaucucucuccgaccTT 394ggucggagagagauccgucTT 395 43 ± 2 AD-14548 gguaccagaucgcgcccau 645-663gguaccagaucgcgcccauTT 396 augggcgcgaucugguaccTT 397 27 ± 3 AD-14549uagguuauuuccgugggau 665-683 uagguuauuuccgugggauTT 398aucccacggaaauaaccuaTT 399 22 ± 1 AD-14550 accuuuugccgggagaccc 767-785accuuuugccgggagacccTT 400 gggucucccggcaaaagguTT 401 89 ± 2 AD-14551ucgcgcucucggcagugcc 830-848 ucgcgcucucggcagugccTT 402ggcacugccgagagcgcgaTT 403 95 ± 3 AD-14552 uauccaccugcaagacuau 956-974uauccaccugcaagacuauTT 404 auagucuugcagguggauaTT 405 15 ± 1 AD-14553ccgcggccagauccugucc 1014-1032 ccgcggccagauccuguccTT 406ggacaggaucuggccgcggTT 407 53 ± 5 AD-14554 aacccacaacgaaaucuau 1212-1230aacccacaacgaaaucuauTT 408 auagauuucguuguggguuTT 409  8 ± 1 AD-14555agcgguaccugaacccgug 1293-1311 agcgguaccugaacccgugTT 410cacggguucagguaccgcuTT 411 14 ± 2 AD-14556 uggcacccagcgacucgcc 1427-1445uggcacccagcgacucgccTT 412 ggcgagucgcugggugccaTT 413 88 ± 9 AD-14557cucgccagagugguuaucu 1440-1458 cucgccagagugguuaucuTT 414agauaaccacucuggcgagTT 415 18 ± 2 AD-14558 cggcagugguugagccgug 1480-1498cggcagugguugagccgugTT 416 cacggcucaaccacugccgTT 417 32 ± 2 AD-14559gugguugagccguggaggg 1485-1503 gugguugagccguggagggTT 418cccuccacggcucaaccacTT 419 24 ± 1 AD-14560 gagggcuuucgccuuagcg 1510-1528gagggcuuucgccuuagcgTT 420 cgcuaaggcgaaagcccucTT 421 51 ± 2 AD-14561agggcuuucgccuuagcgc 1511-1529 agggcuuucgccuuagcgcTT 422gcgcuaaggcgaaagcccuTT 423 37 ± 1 AD-14562 ggugaccuggccaccauuc 1600-1618ggugaccuggccaccauucTT 424 gaaugguggccaggucaccTT 425 51 ± 5 AD-14563accauucauggcaugaacc 1612-1630 accauucauggcaugaaccTT 426gguucaugccaugaaugguTT 427 29 ± 2 AD-14564 cauggcaugaaccggccuu 1618-1636cauggcaugaaccggccuuTT 428 aaggccgguucaugccaugTT 429 25 ± 2 AD-14565ccaacuauugcuucagcuc 1712-1730 ccaacuauugcuucagcucTT 430gagcugaagcaauaguuggTT 431 11 ± 1 AD-14566 gaacugcugcgugcggcag 1740-1758gaacugcugcgugcggcagTT 432 cugccgcacgcagcaguucTT 433 62 ± 3 AD-14567gcccuguacaaccagcaua 1888-1906 gcccuguacaaccagcauaTT 434uaugcugguuguacagggcTT 435 10 ± 0 AD-14568 cuguacaaccagcauaacc 1891-1909cuguacaaccagcauaaccTT 436 gguuaugcugguuguacagTT 437 19 ± 0 AD-14569guacaaccagcauaacccg 1893-1911 guacaaccagcauaacccgTT 438cggguuaugcugguuguacTT 439 21 ± 1 AD-14570 cuauuccuuugcccggcau 2285-2303cuauuccuuugcccggcauTT 440 augccgggcaaaggaauagTT 441 65 ± 2 AD-14571agcggaggaaggagucgcc 143-161 agcggaggaaggagucgccTT 442ggcgacuccuuccuccgcuTT 443 75 ± 2 AD-14572 ggaggaaggagucgccgag 146-164ggaggaaggagucgccgagTT 444 cucggcgacuccuuccuccTT 445 43 ± 2 AD-14573acuuuugagacuuuuccgu 279-297 acuuuugagacuuuuccguTT 446acggaaaagucucaaaaguTT 447 32 ± 2 AD-14574 uugagacuuuuccguugcc 283-301uugagacuuuuccguugccTT 448 ggcaacggaaaagucucaaTT 449 96 ± 4 AD-14575accucuuggcgcgacgcug 324-342 accucuuggcgcgacgcugTT 450cagcgucgcgccaagagguTT 451 67 ± 3 AD-14576 gacccggccucccgcaaag 473-491gacccggccucccgcaaagTT 452 cuuugcgggaggccgggucTT 453 54 ± 3 AD-14577cccggccucccgcaaagac 475-493 cccggccucccgcaaagacTT 454gucuuugcgggaggccgggTT 455 48 ± 4 AD-14578 cuccuccaggagacggauc 579-597cuccuccaggagacggaucTT 456 gauccgucuccuggaggagTT 457 25 ± 1 AD-14579caccuucugguaccagauc 637-655 caccuucugguaccagaucTT 458gaucugguaccagaaggugTT 459 25 ± 1 AD-14580 cuucugguaccagaucgcg 640-658cuucugguaccagaucgcgTT 460 cgcgaucugguaccagaagTT 461 42 ± 2 AD-14581uucugguaccagaucgcgc 641-659 uucugguaccagaucgcgcTT 462gcgcgaucugguaccagaaTT 463 56 ± 2 AD-14582 gguuauuuccgugggauac 667-685gguuauuuccgugggauacTT 464 guaucccacggaaauaaccTT 465 19 ± 2 AD-14583accucagcuuucccucgag 740-758 accucagcuuucccucgagTT 466cucgagggaaagcugagguTT 467 40 ± 4 AD-14584 ccucagcuuucccucgagg 741-759ccucagcuuucccucgaggTT 468 ccucgagggaaagcugaggTT 469 47 ± 5 AD-14585uaccuuuugccgggagacc 766-784 uaccuuuugccgggagaccTT 470ggucucccggcaaaagguaTT 471 102 ± 7  AD-14586 ccaugccgcccuccgggcu 866-884ccaugccgcccuccgggcuTT 472 agcccggagggcggcauggTT 473 54 ± 3 AD-14587acuauccaccugcaagacu 954-972 acuauccaccugcaagacuTT 474agucuugcagguggauaguTT 475 22 ± 2 AD-14588 cuauccaccugcaagacua 955-973cuauccaccugcaagacuaTT 476 uagucuugcagguggauagTT 477 14 ± 1 AD-14589guccaagcugcggcucgcc 1029-1047 guccaagcugcggcucgccTT 478ggcgagccgcagcuuggacTT 479 31 ± 2 AD-14590 cgagccugaggccgacuac 1158-1176cgagccugaggccgacuacTT 480 guagucggccucaggcucgTT 481 16 ± 1 AD-14591ugguggaaacccacaacga 1205-1223 ugguggaaacccacaacgaTT 482ucguuguggguuuccaccaTT 483 98 ± 4 AD-14592 acccacaacgaaaucuaug 1213-1231acccacaacgaaaucuaugTT 484 cauagauuucguuguggguTT 485 14 ± 1 AD-14593ucaagcagaguacacacag 1238-1256 ucaagcagaguacacacagTT 486cuguguguacucugcuugaTT 487 67 ± 8 AD-14594 cagaguacacacagcauau 1243-1261cagaguacacacagcauauTT 488 auaugcuguguguacucugTT 485  9 ± 1 AD-14595ucuucaacacaucagagcu 1268-1286 ucuucaacacaucagagcuTT 490agcucugauguguugaagaTT 491 23 ± 3 AD-14596 gcgguaccugaacccgugu 1294-1312gcgguaccugaacccguguTT 492 acacggguucagguaccgcTT 493 33 ± 2 AD-14597cugcugaggcucaaguuaa 1336-1354 cugcugaggcucaaguuaaTT 494uuaacuugagccucagcagTT 495  8 ± 1 AD-14598 gcgauaccucagcaaccgg 1404-1422gcgauaccucagcaaccggTT 496 ccgguugcugagguaucgcTT 497 15 ± 0 AD-14599ggcacccagcgacucgcca 1428-1446 ggcacccagcgacucgccaTT 498uggcgagucgcugggugccTT 499 20 ± 2 AD-14600 ccagcgacucgccagagug 1433-1451ccagcgacucgccagagugTT 500 cacucuggcgagucgcuggTT 501 17 ± 2 AD-14601gagugguuaucuuuugaug 1447-1465 gagugguuaucuuuugaugTT 502caucaaaagauaaccacucTT 503 13 ± 1 AD-14602 uugugcggcagugguugag 1475-1493uugugcggcagugguugagTT 504 cucaaccacugccgcacaaTT 505 132 ± 9  AD-14603ugugacagcagggauaaca 1540-1558 ugugacagcagggauaacaTT 506uguuaucccugcugucacaTT 507 16 ± 3 AD-14604 acagcagggauaacacacu 1544-1562acagcagggauaacacacuTT 508 aguguguuaucccugcuguTT 509 19 ± 1 AD-14605cagcagggauaacacacug 1545-1563 cagcagggauaacacacugTT 510caguguguuaucccugcugTT 511 34 ± 2 AD-14606 uaacacacugcaaguggac 1554-1572uaacacacugcaaguggacTT 512 guccacuugcaguguguuaTT 513 76 ± 2 AD-14607guucacuaccggccgccga 1581-1599 guucacuaccggccgccgaTT 514ucggcggccgguagugaacTT 515 23 ± 1 AD-14608 uggccaccauucauggcau 1607-1625uggccaccauucauggcauTT 516 augccaugaaugguggccaTT 517 19 ± 1 AD-14609cauucauggcaugaaccgg 1614-1632 cauucauggcaugaaccggTT 518ccgguucaugccaugaaugTT 519 24 ± 2 AD-14610 cagcaucugcaaagcuccc 1672-1690cagcaucugcaaagcucccTT 520 gggagcuuugcagaugcugTT 521 17 ± 2 AD-14611gcaaagcucccggcaccgc 1680-1698 gcaaagcucccggcaccgcTT 522gcggugccgggagcuuugcTT 523 73 ± 5 AD-14612 ggagaagaacugcugcgug 1734-1752ggagaagaacugcugcgugTT 524 cacgcagcaguucuucuccTT 525 13 ± 0 AD-14613gaagaacugcugcgugcgg 1737-1755 gaagaacugcugcgugcggTT 526ccgcacgcagcaguucuucTT 527 62 ± 4 AD-14614 gcuggaaguggauccacga 1787-1805gcuggaaguggauccacgaTT 528 ucguggauccacuuccagcTT 529 12 ± 1 AD-14615uuuggagccuggacacgca 1853-1871 uuuggagccuggacacgcaTT 530ugcguguccaggcuccaaaTT 531 35 ± 1 AD-14616 uuggagccuggacacgcag 1854-1872uuggagccuggacacgcagTT 532 cugcguguccaggcuccaaTT 533 76 ± 7 AD-14617gccgugcugcgugccgcag 1926-1944 gccgugcugcgugccgcagTT 534cugcggcacgcagcacggcTT 535 71 ± 5 AD-14618 uguccaacaugaucgugcg 2003-2021uguccaacaugaucgugcgTT 536 cgcacgaucauguuggacaTT 537 41 ± 2 AD-14619cugcggaucucugugucau 2179-2197 cugcggaucucugugucauTT 538augacacagagauccgcagTT 539 76 ± 7 AD-14620 gcacuauuccuuugcccgg 2282-2300gcacuauuccuuugcccggTT 540 ccgggcaaaggaauagugcTT 541 89 ± 9 AD-14621ccggccucccgcaaagacu 476-494 ccggccucccgcaaagacuTT 542agucuuugcgggaggccggTT 543 48 ± 3 AD-14622 cuacuggugcugacgccug 919-937cuacuggugcugacgccugTT 544 caggcgucagcaccaguagTT 545 14 ± 1 AD-14623auucauggcaugaaccggc 1615-1633 auucauggcaugaaccggcTT 546gccgguucaugccaugaauTT 547 103 ± 6  AD-14624 aacugcugcgugcggcagc1741-1759 aacugcugcgugcggcagcTT 548 gcugccgcacgcagcaguuTT 549 82 ± 3AD-14625 cggaggaaggagucgccga 145-163 cggaggaaggagucgccgaTT 550ucggcgacuccuuccuccgTT 551 47 ± 3 AD-14626 ggagcgggaggagggacga 236-254ggagcgggaggagggacgaTT 552 ucgucccuccucccgcuccTT 553 31 ± 1 AD-14627gcccucgggagucgccgac 457-475 gcccucgggagucgccgacTT 554gucggcgacucccgagggcTT 555 56 ± 6 AD-14628 uuucuccuccaggagacgg 576-594uuucuccuccaggagacggTT 556 ccgucuccuggaggagaaaTT 557 90 ± 3 AD-14629ucgaggcccuccuaccuuu 754-772 ucgaggcccuccuaccuuuTT 558aaagguaggagggccucgaTT 559 25 ± 2 AD-14630 uacuggugcugacgccugg 920-938uacuggugcugacgccuggTT 560 ccaggcgucagcaccaguaTT 561 34 ± 2 AD-14631gacuauccaccugcaagac 953-971 gacuauccaccugcaagacTT 562gucuugcagguggauagucTT 563 24 ± 3 AD-14632 uacaacagcacccgcgacc 1108-1126uacaacagcacccgcgaccTT 564 ggucgcgggugcuguuguaTT 565 113 ± 4  AD-14633ugcugaggcucaaguuaaa 1337-1355 ugcugaggcucaaguuaaaTT 566uuuaacuugagccucagcaTT 567  8 ± 1 AD-14634 ccugugacagcagggauaa 1538-1556ccugugacagcagggauaaTT 568 uuaucccugcugucacaggTT 569 14 ± 1 AD-14635auaacacacugcaagugga 1553-1571 auaacacacugcaaguggaTT 570uccacuugcaguguguuauTT 571 18 ± 1 AD-14636 uauugcuucagcuccacgg 1717-1735uauugcuucagcuccacggTT 572 ccguggagcugaagcaauaTT 573 109 ± 5  AD-14637gcuguccaacaugaucgug 2001-2019 gcuguccaacaugaucgugTT 574cacgaucauguuggacagcTT 575 29 ± 1 AD-14638 gucuccaucccugacguuc 2214-2232gucuccaucccugacguucTT 576 gaacgucagggauggagacTT 577  75 ± 11 ¹a, g, c,u: ribonucleotide 5′-monophosphates (except where in 5′-most position,see below); T: deoxythymidine 5′-monophosphate; all sequences given5′ → 3′; due to synthesis procedures, the 5′-most nucleic acid is anucleoside, i.e. does not bear a 5′-monophosphate group

1. An isolated iRNA agent, comprising an antisense strand and a sensestrand complementary to said antisense strand, wherein the antisensestrand comprises SEQ ID NO:88 (cmaaugumacmagcugccgcmacTT*), wherein cmis 2′-O-methylcytidine-5′-monophosphate, wherein um is2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine5′-monothiophosphate.
 2. The iRNA agent of claim 1, wherein said iRNAagent mediates the cleavage of a TGF-beta mRNA within the targetsequence iRNA agent AL-DP-6858.
 3. The iRNA agent of claim 1, whereinthe iRNA agent further comprises a non-nucleotide moiety.
 4. The iRNA ofclaim 1, wherein the sense and/or antisense strand is further stabilizedagainst nucleolytic degradation.
 5. The iRNA agent of claim 1, whereinthe sense strand further comprises at least one 3′-overhang wherein said3′-overhang comprises from 1 to 6 nucleotides.
 6. The iRNA agent ofclaim 1, further comprising a phosphorothioate at the firstinternucleotide linkage at the 5′ end of the sense strand.
 7. The iRNAagent of claim 6, further comprising a phosphorothioate at the firstinternucleotide linkage at the 3′ end of the sense strand.
 8. The iRNAagent of claim 1, further comprising a 2′-modified nucleotide in thesense strand.
 9. The iRNA agent of claim 8, wherein the 2′-modifiednucleotide comprises a modification selected from the group consistingof: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O—NMA).
 10. The iRNA agent of claim 1,wherein said sense strand is 21 contiguous nucleotides of the sensesequence of iRNA agent AL-DP-6858 (SEQ ID NO:85).
 11. The iRNA agent ofclaim 1, wherein said sense strand comprises SEQ ID NO:85.
 12. The iRNAagent of claim 1, wherein said sense strand consists of SEQ ID NO:85.13. The iRNA agent of claim 1, wherein the iRNA agent is AL-DP-6271. 14.The iRNA agent of claim 1, wherein the antisense consists of SEQ IDNO:88 (cmaaugumacmagcugccgcmacTT*), wherein cm is2′-O-methylcytidine-5′-monophosphate, wherein um is2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine5′-monothiophosphate.
 15. The iRNA agent of claim 1, wherein the iRNAagent comprises AL-DP-6271.
 16. An isolated iRNA agent, comprising anantisense strand and a sense strand complementary to said antisensestrand, wherein the sense strand comprises SEQ ID NO:87(gumgcmggcmagcmumgumacmaumumgTT*), wherein cm is2′-O-methylcytidine-5′-monophosphate, wherein um is2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine5′-monothiophosphate.
 17. The iRNA agent of claim 16, wherein the sensestrand consists of SEQ ID NO:87 (gumgcmggcmagcmumgumacmaumumgTT*),wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine5′-monothiophosphate.
 18. An isolated iRNA agent, comprising anantisense strand and a sense strand complementary to said antisensestrand, wherein the sense strand comprises SEQ ID NO:87(gumgcmggcmagcmumgumacmaumumgTT*) and the antisense strand comprises SEQID NO:88 (cmaaugumacmagcugccgcmacTT*), wherein cm is2′-O-methylcytidine-5′-monophosphate, wherein um is2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine5′-monothiophosphate.
 19. The iRNA agent of claim 18, wherein the sensestrand consists of SEQ ID NO:87 (gumgcmggcmagcmumgumacmaumumgTT*) andthe antisense strand consists of SEQ ID NO:88(cmaaugumacmagcugccgcmacTT*), wherein cm is2′-O-methylcytidine-5′-monophosphate, wherein um is2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine5′-monothiophosphate.
 20. A vector encoding the iRNA agent of claim 1.21. A cell comprising the iRNA agent of claim 1 or the vector of claim20.
 22. A method of making the iRNA agent of claim 1, the methodcomprising the synthesis of the iRNA agent, wherein the sense andantisense strands comprise at least one modification that stabilizes theiRNA agent against nucleolytic degradation.
 23. A pharmaceuticalcomposition comprising the iRNA agent of claim 1 and a pharmaceuticallyacceptable carrier.
 24. A method of treating a human diagnosed as havinga disease or disorder associated with undesired TGF-beta signaling,comprising administering to a subject in need of such treatment atherapeutically effective amount of the iRNA agent of claim
 1. 25. Themethod of claim 24, wherein the human is diagnosed as having idiopathicpulmonary fibrosis, diabetic nephropathy or chronic liver disease.
 26. Amethod of reducing the levels of a TGF-beta mRNA in a cell of a subject,or of TGF-beta protein secreted by a cell of a subject, comprising thestep of administering the iRNA agent of claim 1 to said subject.
 27. Themethod of claim 26 wherein said iRNA agent mediates the cleavage of aTGF-beta mRNA within the target sequence of iRNA agent AL-DP-6858. 28.The method of claim 26, wherein said iRNA agent is administered to thesubject.
 29. The method of claim 28, wherein said administrationcomprises pulmonary administration.
 30. The method of claim 26, whereinthe level of TGF-beta protein and/or TGF-beta mRNA is reduced by atleast 20%.
 31. The method of claim 26, wherein said sense strand is 21contiguous nucleotides of the sense sequence of iRNA agent AL-DP-6858(SEQ ID NO:85).
 32. A method of reducing the levels of a TGF-beta mRNAin a cell, or of TGF-beta protein secreted by a cell, comprisingcontacting the cell with the iRNA agent of claim 1.